Methods of treating locally advanced or metastatic breast cancers using pd-1 axis binding antagonists and taxanes

ABSTRACT

The invention provides methods and compositions for treating locally advanced or metastatic breast cancer and for enhancing immune function in an individual having locally advanced or metastatic breast cancer. The methods comprise administering a PD-1 axis binding antagonist and a taxane.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 6, 2021, isnamed 50474-119003_Sequence_Listing_08_06_21_ST25 and is 23,503 bytes insize.

FIELD OF THE INVENTION

This invention relates to methods of treating locally advanced ormetastatic breast cancers and for enhancing immune function in anindividual having locally advanced or metastatic breast cancer byadministering a PD-1 axis binding antagonist and a taxane.

BACKGROUND OF THE INVENTION

The provision of two distinct signals to T-cells is a widely acceptedmodel for lymphocyte activation of resting T lymphocytes byantigen-presenting cells (APCs). This model further provides for thediscrimination of self from non-self and immune tolerance. The primarysignal, or antigen-specific signal, is transduced through the T-cellreceptor (TCR) following recognition of foreign antigen peptidepresented in the context of the major histocompatibility-complex (MHC).The second signal, or co-stimulatory signal, is delivered to T-cells byco-stimulatory molecules expressed on APCs and induces T-cells topromote clonal expansion, cytokine secretion and effector function. Inthe absence of co-stimulation, T-cells can become refractory to antigenstimulation, which results in a tolerogenic response to either foreignor endogenous antigens.

In the two-signal model, T-cells receive both positive and negativesecondary co-stimulatory signals. The regulation of such positive andnegative signals is critical to maximize the host's protective immuneresponses, while maintaining immune tolerance and preventingautoimmunity. Negative secondary signals seem necessary for induction ofT-cell tolerance, while positive signals promote T-cell activation.While the simple two-signal model still provides a valid explanation fornaïve lymphocytes, a host's immune response is a dynamic process, andco-stimulatory signals can also be provided to antigen-exposed T-cells.

The mechanism of co-stimulation is of therapeutic interest because themanipulation of co-stimulatory signals provides a means to eitherenhance or terminate cell-based immune response. T cell dysfunction oranergy occurs concurrently with an induced and sustained expression ofthe inhibitory receptor, programmed death 1 polypeptide (PD-1), whichbinds to ligands that include PD-L1 and PD-L2. PD-L1 is overexpressed inmany cancers and is often associated with poor prognosis. The majorityof tumor-infiltrating T lymphocytes predominantly express PD-1, incontrast to T lymphocytes in normal tissues and peripheral blood Tlymphocytes, indicating that up-regulation of PD-1 on tumor-reactive Tcells can contribute to impaired anti-tumor immune responses. This maybe due to exploitation of PD-L1 signaling mediated by PD-L1-expressingtumor cells interacting with PD-1-expressing T cells, resulting inattenuation of T cell activation and evasion of immune surveillance.Therefore, inhibition of the PD-L1/PD-1 interaction may enhance CD8+ Tcell-mediated killing of tumors.

An optimal therapeutic treatment may combine blockade of PD-1receptor/ligand interaction with an agent that directly inhibits tumorgrowth. There remains a need for an optimal therapy for treating,stabilizing, preventing, and/or delaying development of various cancers(e.g., locally advanced or metastatic breast, e.g., metastatictriple-negative breast cancer (mTNBC)).

SUMMARY OF THE INVENTION

This invention relates to methods of treating locally advanced ormetastatic breast cancers (e.g., metastatic triple-negative breastcancer (mTNBC)) and for enhancing immune function in an individualhaving locally advanced or metastatic breast cancer by administering aPD-1 axis binding antagonist and a taxane.

In one aspect, the invention features a method for treating or delayingprogression of locally advanced or metastatic breast cancer (e.g.,mTNBC) in an individual comprising administering to the individual aneffective amount of a human PD-1 axis binding antagonist and a taxane.In some embodiments, the PD-1 axis binding antagonist is selected fromthe group consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist, and a PD-L2 binding antagonist.

In some embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-1 binding antagonist. In some embodiments, the PD-1binding antagonist inhibits the binding of PD-1 to its ligand bindingpartners. In some embodiments, the PD-1 binding antagonist inhibits thebinding of PD-1 to PD-L1. In some embodiments, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L2. In some embodiments,the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1and PD-L2. In some embodiments, the PD-1 binding antagonist is anantibody. In some embodiments, the PD-1 binding antagonist is selectedfrom the group consisting of MDX 1106 (nivolumab), MK-3475(pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001,REGN2810, and BGB-108.

In other embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1binding antagonist inhibits the binding of PD-L1 to PD-1. In someembodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits thebinding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1binding antagonist is an antibody. In some embodiments, the antibody isselected from the group consisting of: MPDL3280A (atezolizumab),YW243.55.S70, MDX-1105, MED14736 (durvalumab), and MSB0010718C(avelumab). In some embodiments, the antibody is MPDL3280A. In someembodiments, MPDL3280A is administered at a dose of about 700 mg toabout 900 mg every two weeks (e.g., about 750 mg to about 900 mg everytwo weeks, e.g., about 800 mg to about 850 mg every two weeks). In someembodiments, MPDL3280A is administered at a dose of about 840 mg everytwo weeks.

In some embodiments, the antibody comprises a heavy chain comprising anHVR-H1 sequence of SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, andHVR-H3 sequence of SEQ ID NO:21; and a light chain comprising an HVR-L1sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3sequence of SEQ ID NO:24. In some embodiments, the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:25 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:4.

In some embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2binding antagonist is an antibody. In some embodiments, the PD-L2binding antagonist is an immunoadhesin.

In any of the preceding embodiments of the above aspect, the locallyadvanced or metastatic breast cancer may be, without limitation,triple-negative breast cancer (TNBC) or metastatic triple-negativebreast cancer (mTNBC).

In any of the preceding embodiments of the above aspect, the individualhas locally advanced or metastatic breast cancer or has been diagnosedwith locally advanced or metastatic breast cancer. In some embodiments,the cancer cells in the individual express PD-L1. In some embodiments,the expression of PD-L1 may be determined by an immunohistochemistry(IHC) assay.

In any of the preceding embodiments of the above aspect, the individualhas had two or fewer prior cytotoxic treatment regimens (e.g., 0, 1, or2 prior cytotoxic treatment regimen(s)) for locally advanced ormetastatic breast cancer.

In any of the preceding embodiments of the above aspect, the individualhas never had prior systemic treatment for locally advanced ormetastatic breast cancer (e.g., mTNBC). Accordingly, in any of themethods described herein, the administration of an effective amount of ahuman PD-1 axis binding antagonist and a taxane may be a first-linetherapy for locally advanced or metastatic breast cancer.

In any of the preceding embodiments of the above aspect, the treatmentmay result in a response in the individual. In some embodiments, theresponse is a complete response. In some embodiments, the response is asustained response after cessation of the treatment. In someembodiments, the response is a complete response that is sustained aftercessation of the treatment. In other embodiments, the response is apartial response. In some embodiments, the response is a partialresponse that is sustained after cessation of the treatment.

In any of the preceding embodiments of the above aspect, the taxane isadministered before the PD-1 axis binding antagonist, simultaneous withthe PD-1 axis binding antagonist, or after the PD-1 axis bindingantagonist. In some embodiments, the taxane is nab-paclitaxel(ABRAXANE®), paclitaxel, or docetaxel. In some embodiments, the taxaneis nab-paclitaxel (ABRAXANE®). In some embodiments, the nab-paclitaxel(ABRAXANE®) is administered to the individual at a dose of about 50mg/m² to about 175 mg/m² every week (e.g., about 75 mg/m² to about 150mg/m² every week, e.g., about 80 mg/m² to about 125 mg/m² every week,e.g., about 100 mg/m² to about 125 mg/m² every week). In someembodiments, the nab-paclitaxel (ABRAXANE®) is administered to theindividual at a dose of about 100 mg/m² every week. In some embodiments,the taxane is paclitaxel.

In another aspect, the invention features a method for treating ordelaying progression of locally advanced or metastatic breast cancer inan individual, wherein the method comprises a dosing regimen comprisingtreatment cycles, wherein the individual is administered, on days 1 and15 of each cycle, a human PD-1 axis binding antagonist at a dose ofabout 840 mg, and on days 1, 8, and 15 of each cycle, a taxane at a doseof about 100 mg/m², each cycle being repeated every 28 days. In someembodiments, the cycle is repeated until there is loss of clinicalbenefit. In some embodiments, cycles are repeated in which only the PD-1axis binding antagonist is administered. In some embodiments, themetastatic breast cancer is mTNBC. In some embodiments, the PD-1 axisbinding antagonist is a PD-L1 binding antagonist. In some embodiments,the PD-L1 binding antagonist is MPDL3280A. In some embodiments, thetaxane is nab-paclitaxel (ABRAXANE®). In some embodiments, theindividual has had two or fewer prior cytotoxic treatment regimens forlocally advanced or metastatic breast cancer. In some embodiments, theindividual has never had prior targeted systemic treatment for locallyadvanced or metastatic breast cancer.

In another aspect, the invention features a method of enhancing immunefunction in an individual having locally advanced or metastatic breastcancer, the method comprising administering an effective amount of aPD-1 axis binding antagonist and a taxane. In some embodiments, CD8+ Tcells in the individual have enhanced priming, activation,proliferation, and/or cytolytic activity relative to prior to theadministration of the PD-1 axis binding antagonist and the taxane. Insome embodiments, the number of CD8+ T cells is elevated relative toprior to administration of the combination. In some embodiments, theCD8+ T cell is an antigen-specific CD8+ T cell. In some embodiments,Treg function is suppressed relative to prior to the administration ofthe combination. In some embodiments, T cell exhaustion is decreasedrelative to prior to the administration of the combination. In someembodiments, the PD-1 axis binding antagonist is selected from the groupconsisting of a PD-1 binding antagonist, a PD-L1 binding antagonist anda PD-L2 binding antagonist.

In some embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-1 binding antagonist. In some embodiments, the PD-1binding antagonist inhibits the binding of PD-1 to its ligand bindingpartners. In some embodiments, the PD-1 binding antagonist inhibits thebinding of PD-1 to PD-L1. In some embodiments, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L2. In some embodiments,the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1and PD-L2. In some embodiments, the PD-1 binding antagonist is anantibody. In some embodiments, the PD-1 binding antagonist is selectedfrom the group consisting of MDX 1106 (nivolumab), MK-3475(pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001,REGN2810, and BGB-108.

In other embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1binding antagonist inhibits the binding of PD-L1 to PD-1. In someembodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits thebinding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1binding antagonist is an antibody. In some embodiments, the antibody isselected from the group consisting of: MPDL3280A (atezolizumab),YW243.55.S70, MDX-1105, MED14736 (durvalumab), and MSB0010718C(avelumab). In some embodiments, the antibody is MPDL3280A. In someembodiments, MPDL3280A is administered at a dose of about 700 mg toabout 900 mg every two weeks (e.g., about 750 mg to about 900 mg everytwo weeks, e.g., about 800 mg to about 850 mg every two weeks). In someembodiments, MPDL3280A is administered at a dose of about 840 mg everytwo weeks.

In some embodiments, the antibody comprises a heavy chain comprising anHVR-H1 sequence of SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, andHVR-H3 sequence of SEQ ID NO:21; and a light chain comprising an HVR-L1sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3sequence of SEQ ID NO:24. In some embodiments, the antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:25 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:4.

In other embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2binding antagonist is an antibody. In some embodiments, the PD-L2binding antagonist is an immunoadhesin.

In any of the preceding embodiments of the above aspect, the locallyadvanced or metastatic breast cancer may be, without limitation,triple-negative breast cancer (TNBC) or metastatic triple-negativebreast cancer (mTNBC).

In some embodiments, the cancer cells in the individual express PD-L1.In some embodiments, the expression of PD-L1 may be determined by animmunohistochemistry (IHC) assay.

In any of the preceding embodiments of the above aspect, the individualhas had two or fewer prior cytotoxic treatment regimens (e.g., 0, 1, or2 prior cytotoxic treatment regimen(s)) for locally advanced ormetastatic breast cancer.

In any of the preceding embodiments of the above aspect, the individualhas never had prior systemic treatment for locally advanced ormetastatic breast cancer (e.g., mTNBC).

In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®),paclitaxel, or docetaxel. In some embodiments, the taxane isnab-paclitaxel (ABRAXANE®). In some embodiments, the nab-paclitaxel(ABRAXANE®) is administered to the individual at a dose of about 50mg/m² to about 175 mg/m² every week (e.g., about 75 mg/m² to about 150mg/m² every week, e.g., about 80 mg/m² to about 125 mg/m² every week,e.g., about 100 mg/m² to about 125 mg/m² every week). In someembodiments, the nab-paclitaxel (ABRAXANE®) is administered to theindividual at a dose of about 100 mg/m² every week. In some embodiments,the taxane is paclitaxel.

In another aspect, the invention features a method of enhancing immunefunction in an individual having locally advanced or metastatic breastcancer, wherein the method comprises a dosing regimen comprisingtreatment cycles, wherein the individual is administered, on days 1 and15 of each cycle, a human PD-1 axis binding antagonist at a dose ofabout 840 mg, and on days 1, 8, and 15 of each cycle, a taxane at a doseof about 100 mg/m², each cycle being repeated every 28 days. In someembodiments, the CD8+ T cells in the individual have enhanced priming,activation, proliferation, and/or cytolytic activity relative to priorto the administration of the PD-1 axis binding antagonist and thetaxane. In some embodiments, the number of CD8+ T cells is elevatedrelative to prior to administration of the combination. In someembodiments, the CD8+ T cell is an antigen-specific CD8+ T cell. In someembodiments, Treg function is suppressed relative to prior to theadministration of the combination. In some embodiments, T cellexhaustion is decreased relative to prior to the administration of thecombination. In some embodiments, the PD-1 axis binding antagonist isselected from the group consisting of a PD-1 binding antagonist, a PD-L1binding antagonist and a PD-L2 binding antagonist.

In some embodiments, the locally advanced or metastatic breast cancermay be, without limitation, TNBC or mTNBC. In some embodiments, the PD-1axis binding antagonist is a PD-L1 binding antagonist. In someembodiments, the PD-L1 binding antagonist is MPDL3280A. In someembodiments, the taxane is nab-paclitaxel (ABRAXANE®). In someembodiments, the individual has had two or fewer prior cytotoxictreatment regimens for locally advanced or metastatic breast cancer. Insome embodiments, the individual has never had prior targeted systemictreatment for locally advanced or metastatic breast cancer.

In some embodiments of any one of the above aspects, the PD-1 axisbinding antagonist and/or the taxane are administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally.

In some embodiments of any one of the above aspects, the method mayfurther comprise administering an effective amount of a chemotherapeuticagent.

In another aspect, the invention features a use of a human PD-1 axisbinding antagonist in the manufacture of a medicament for treating ordelaying progression of a locally advanced or metastatic breast cancerin an individual, wherein the medicament comprises the human PD-1 axisbinding antagonist and an optional pharmaceutically acceptable carrier,and wherein the treatment comprises administration of the medicament incombination with a composition comprising a taxane and an optionalpharmaceutically acceptable carrier. In some embodiments, the PD-1 axisbinding antagonist is a PD-L1 binding antagonist. In some embodiments,the PD-L1 binding antagonist is MPDL3280A. In some embodiments, themedicament comprises MPDL3280A at a dose of about 840 mg. In someembodiments, the treatment comprises administration of the medicamentonce every two weeks to the individual. In some embodiments, the taxaneis nab-paclitaxel (ABRAXANE®). In some embodiments, the treatmentcomprises administration of the composition to the individual at a doseof about 100 mg/m² of nab-paclitaxel (ABRAXANE®). In some embodiments,the treatment comprises administration of the composition once everyweek to the individual. In some embodiments, the medicament comprisesMPDL3280A at a dose of about 840 mg, taxane is nab-paclitaxel(ABRAXANE®), and the treatment comprises administration of themedicament once every two weeks to the individual and administration ofthe composition once every week to the individual at a dose of about 100mg/m² of nab-paclitaxel (ABRAXANE®). In some embodiments, the metastaticbreast cancer is mTNBC.

In another aspect, the invention features a use of a taxane in themanufacture of a medicament for treating or delaying progression of alocally advanced or metastatic breast cancer in an individual, whereinthe medicament comprises the taxane and an optional pharmaceuticallyacceptable carrier, and wherein the treatment comprises administrationof the medicament in combination with a composition comprising a humanPD-1 axis binding antagonist and an optional pharmaceutically acceptablecarrier. In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®).In some embodiments, the treatment comprises administration of themedicament to the individual at a dose of about 100 mg/m² ofnab-paclitaxel (ABRAXANE®). In some embodiments, the treatment comprisesadministration of the medicament once every week to the individual. Insome embodiments, the PD-1 axis binding antagonist is a PD-L1 bindingantagonist. In some embodiments, the PD-L1 binding antagonist isMPDL3280A. In some embodiments, the composition comprises MPDL3280A at adose of about 840 mg. In some embodiments, the treatment comprisesadministration of the composition once every two weeks to theindividual. In some embodiments, the medicament comprises nab-paclitaxel(ABRAXANE®) at a dose of about 100 mg/m² of nab-paclitaxel (ABRAXANE®),the composition comprises MPDL3280A at a dose of about 840 mg, and thetreatment comprises administration of the medicament once every week andadministration of the composition once every two weeks to theindividual. In some embodiments, the metastatic breast cancer is mTNBC.

In another aspect, the invention features a composition comprising ahuman PD-1 axis binding antagonist and an optional pharmaceuticallyacceptable carrier for use in treating or delaying progression of alocally advanced or metastatic breast cancer in an individual, whereinthe treatment comprises administration of said composition incombination with a second composition, wherein the second compositioncomprises a taxane and an optional pharmaceutically acceptable carrier.In some embodiments, the PD-1 axis binding antagonist is a PD-L1 bindingantagonist. In some embodiments, the PD-L1 binding antagonist isMPDL3280A. In some embodiments, the composition comprises MPDL3280A at adose of about 840 mg. In some embodiments, the treatment comprisesadministration of the composition once every two weeks to theindividual. In some embodiments, the taxane is nab-paclitaxel(ABRAXANE®). In some embodiments, the second composition comprisesnab-paclitaxel (ABRAXANE®) at a dose of about 100 mg/m². In someembodiments, the treatment comprises administration of the secondcomposition once every week to the individual. In some embodiments, thecomposition comprises MPDL3280A at a dose of about 840 mg, the secondcomposition comprises nab-paclitaxel (ABRAXANE®) at a dose of about 100mg/m², and the treatment comprises administration of the compositiononce every two weeks and administration of the second composition onceevery week to the individual.

In another aspect, the invention features a composition comprising ataxane and an optional pharmaceutically acceptable carrier for use intreating or delaying progression of a locally advanced or metastaticbreast cancer in an individual, wherein the treatment comprisesadministration of said composition in combination with a secondcomposition, wherein the second composition comprises a human PD-1 axisbinding antagonist and an optional pharmaceutically acceptable carrier.In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In someembodiments, the composition comprises nab-paclitaxel (ABRAXANE®) at adose of about 100 mg/m². In some embodiments, the treatment comprisesadministration of the composition once every week to the individual. Insome embodiments, the PD-1 axis binding antagonist is a PD-L1 bindingantagonist. In some embodiments, the PD-L1 binding antagonist isMPDL3280A. In some embodiments, the second composition comprisesMPDL3280A at a dose of about 840 mg. In some embodiments, the treatmentcomprises administration of the second composition once every two weeksto the individual. In some embodiments, the composition comprisesnab-paclitaxel (ABRAXANE®) at a dose of about 100 mg/m², the secondcomposition comprises MPDL3280A at a dose of about 840 mg, and thetreatment comprises administration of the composition once every weekand administration of the second composition once every two weeks to theindividual.

In another aspect, the invention features a kit comprising a medicamentcomprising a PD-1 axis binding antagonist and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the medicament in combination with acomposition comprising a taxane and an optional pharmaceuticallyacceptable carrier for treating or delaying progression of a locallyadvanced or metastatic breast cancer in an individual. In someembodiments, the PD-1 axis binding antagonist is a PD-L1 bindingantagonist. In some embodiments, the PD-L1 binding antagonist isMPDL3280A. In some embodiments, the medicament comprises MPDL3280A at adose of about 840 mg. In some embodiments, the package insert comprisesinstructions for administration of the medicament once every two weeksto the individual. In some embodiments, the taxane is nab-paclitaxel(ABRAXANE®). In some embodiments, the composition comprisesnab-paclitaxel (ABRAXANE®) at a dose of about 100 mg/m². In someembodiments, the package insert comprises instructions foradministration of the composition once every week to the individual. Insome embodiments, the medicament comprises MPDL3280A at a dose of about840 mg, the composition comprises nab-paclitaxel (ABRAXANE®) at a doseof about 100 mg/m², and the package insert comprises instructions foradministration of the medicament once every two weeks and administrationof the composition once every week to the individual.

In another aspect, the invention features a kit comprising a firstmedicament comprising a PD-1 axis binding antagonist and an optionalpharmaceutically acceptable carrier, and a second medicament comprisinga taxane and an optional pharmaceutically acceptable carrier. In someembodiments, the kit further comprises a package insert comprisinginstructions for administration of the first medicament and the secondmedicament for treating or delaying progression of a locally advanced ormetastatic breast cancer in an individual. In some embodiments, the PD-1axis binding antagonist is a PD-L1 binding antagonist. In someembodiments, the PD-L1 binding antagonist is MPDL3280A. In someembodiments, the first medicament comprises MPDL3280A at a dose of about840 mg. In some embodiments, the package insert comprises instructionsfor administration of the first medicament once every two weeks to theindividual. In some embodiments, the taxane is nab-paclitaxel(ABRAXANE®). In some embodiments, the second medicament comprisesnab-paclitaxel (ABRAXANE®) at a dose of about 100 mg/m². In someembodiments, the package insert comprises instructions foradministration of the second medicament once every week to theindividual. In some embodiments, the first medicament comprisesMPDL3280A at a dose of about 840 mg, the second medicament comprisesnab-paclitaxel (ABRAXANE®) at a dose of about 100 mg/m², and the packageinsert comprises instructions for administration of the first medicamentonce every two weeks and administration of the second medicament onceevery week to the individual.

In another aspect, the invention features a kit comprising a medicamentcomprising a taxane and an optional pharmaceutically acceptable carrier,and a package insert comprising instructions for administration of themedicament in combination with a composition comprising a PD-1 axisbinding antagonist and an optional pharmaceutically acceptable carrierfor treating or delaying progression of a locally advanced or metastaticbreast cancer in an individual. In some embodiments, the taxane isnab-paclitaxel (ABRAXANE®). In some embodiments, the medicamentcomprises nab-paclitaxel (ABRAXANE®) at a dose of about 100 mg/m². Insome embodiments, the package insert comprises instructions foradministration of the medicament once every week to the individual. Insome embodiments, the PD-1 axis binding antagonist is a PD-L1 bindingantagonist. In some embodiments, the PD-L1 binding antagonist isMPDL3280A. In some embodiments, the composition comprises MPDL3280A at adose of about 840 mg. In some embodiments, the package insert comprisesinstructions for administration of the composition once every two weeksto the individual. In some embodiments, the medicament comprisesnab-paclitaxel (ABRAXANE®) at a dose of about 100 mg/m², the compositioncomprises MPDL3280A at a dose of about 840 mg, and the package insertcomprises instructions for administration of the medicament once everyweek and administration of the composition once every two weeks to theindividual.

In any one of the preceding aspects, the PD-1 axis binding antagonistmay be selected from the group consisting of a PD-1 binding antagonist,a PD-L1 binding antagonist, and a PD-L2 binding antagonist. In someembodiments, the PD-1 axis binding antagonist is a PD-1 bindingantagonist. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to its ligand binding partners. In some embodiments,the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. Insome embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, thePD-1 binding antagonist is an antibody. In some embodiments, the PD-1binding antagonist is selected from the group consisting of MDX 1106(nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680(AMP-514), PDR001, REGN2810, and BGB-108. In some embodiments, the PD-1axis binding antagonist is a PD-L1 binding antagonist. In someembodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits thebinding of PD-L1 to B7-1. In some embodiments, the PD-L1 bindingantagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In someembodiments, the PD-L1 binding antagonist is an antibody. In someembodiments, the antibody is selected from the group consisting of:MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MED14736 (durvalumab),and MSB0010718C (avelumab). In some embodiments, the antibody comprisesa heavy chain comprising an HVR-H1 sequence of SEQ ID NO:19, HVR-H2sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and alight chain comprising an HVR-L1 sequence of SEQ ID NO:22, HVR-L2sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24. In someembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:25 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:4.

In any one of the preceding aspects, the taxane may be nab-paclitaxel(ABRAXANE®), paclitaxel, or docetaxel. In some embodiments, the taxaneis nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane ispaclitaxel.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partner, so as to remove T-cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T-cell function (e.g., proliferation, cytokineproduction, and/or target cell killing). As used herein, a PD-1 axisbinding antagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist, and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1 and/or PD-L2. In some embodiments, the PD-1binding antagonist is a molecule that inhibits the binding of PD-1 toone or more of its binding partners. In a specific aspect, the PD-1binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2.For example, PD-1 binding antagonists include anti-PD-1 antibodies,antigen-binding fragments thereof, immunoadhesins, fusion proteins,oligopeptides, and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T-cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist isMDX-1106 (nivolumab) described herein. In another specific aspect, aPD-1 binding antagonist is MK-3475 (pembrolizumab) described herein. Inanother specific aspect, a PD-1 binding antagonist is CT-011(pidilizumab) described herein. In another specific aspect, a PD-1binding antagonist is MEDI-0680 (AMP-514) described herein. In anotherspecific aspect, a PD-1 binding antagonist is PDR001 described herein.In another specific aspect, a PD-1 binding antagonist is REGN2810described herein. In another specific aspect, a PD-1 binding antagonistis BGB-108 described herein.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1 and/or B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1 and/or B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is MPDL3280A (atezolizumab, marketed as TECENTRIQ™with a WHO Drug Information (International Nonproprietary Names forPharmaceutical Substances), Recommended INN: List 74, Vol. 29, No. 3,2015 (see page 387)) described herein. In another specific aspect, ananti-PD-L1 antibody is MDX-1105 described herein. In still anotherspecific aspect, an anti-PD-L1 antibody is YW243.55.570 describedherein. In still another specific aspect, an anti-PD-L1 antibody isMED14736 (durvalumab) described herein. In still another specificaspect, an anti-PD-L1 antibody is MSB0010718C (avelumab) describedherein.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific aspect, the PD-L2 bindingantagonist inhibits binding of PD-L2 to PD-1. In some embodiments, thePD-L2 antagonists include anti-PD-L2 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L2 witheither one or more of its binding partners, such as PD-1. In oneembodiment, a PD-L2 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L2 so as rendera dysfunctional T-cell less dysfunctional (e.g., enhancing effectorresponses to antigen recognition). In some embodiments, a PD-L2 bindingantagonist is an immunoadhesin.

A “taxane” as used herein is a diterpene which may bind to tubulin,promoting microtubule assembly and stabilization and/or preventmicrotubule depolymerization. Taxanes included herein include taxoid10-deacetylbaccatin III and/or derivatives thereof. Exemplary taxanesinclude, but are not limited to, paclitaxel (i.e., TAXOL®, CAS#33069-62-4), docetaxel (i.e., TAXOTERE®, CAS #114977-28-5), larotaxel,cabazitaxel, milataxel, tesetaxel, and/or orataxel. In some embodiments,the taxane is an albumin-coated nanoparticle (e.g., nano-albumin bound(nab)-paclitaxel, i.e., ABRAXANE® and/or nab-docetaxel, ABI-008). Insome embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In someembodiments, the taxane is formulated in CREMAPHOR® (e.g., TAXOL®)and/or in Tween such as polysorbate 80 (e.g., TAXOTERE®). In someembodiments, the taxane is liposome-encapsulated taxane. In someembodiments, the taxane is a prodrug form and/or conjugated form oftaxane (e.g., DHA covalently conjugated to paclitaxel, paclitaxelpoliglumex, and/or linoleyl carbonate-paclitaxel). In some embodiments,the paclitaxel is formulated with substantially no surfactant (e.g., inthe absence of CREMAPHOR and/or Tween-such as TOCOSOL® paclitaxel).

The term “dysfunction” in the context of immune dysfunction, refers to astate of reduced immune responsiveness to antigenic stimulation. Theterm includes the common elements of both “exhaustion” and/or “anergy”in which antigen recognition may occur, but the ensuing immune responseis ineffective to control infection or tumor growth.

The term “dysfunctional,” as used herein, also includes refractory orunresponsive to antigen recognition, specifically, impaired capacity totranslate antigen recognition into down-stream T-cell effectorfunctions, such as proliferation, cytokine production (e.g., IL-2)and/or target cell killing.

The term “anergy” refers to the state of unresponsiveness to antigenstimulation resulting from incomplete or insufficient signals deliveredthrough the T-cell receptor (e.g., increase in intracellular Ca⁺² in theabsence of ras-activation). T cell anergy can also result uponstimulation with antigen in the absence of co-stimulation, resulting inthe cell becoming refractory to subsequent activation by the antigeneven in the context of co-stimulation. The unresponsive state can oftenbe overriden by the presence of Interleukin-2. Anergic T-cells do notundergo clonal expansion and/or acquire effector functions.

The term “exhaustion” refers to T cell exhaustion as a state of T celldysfunction that arises from sustained TCR signaling that occurs duringmany chronic infections and cancer. It is distinguished from anergy inthat it arises not through incomplete or deficient signaling, but fromsustained signaling. It is defined by poor effector function, sustainedexpression of inhibitory receptors and a transcriptional state distinctfrom that of functional effector or memory T cells. Exhaustion preventsoptimal control of infection and tumors. Exhaustion can result from bothextrinsic negative regulatory pathways (e.g., immunoregulatorycytokines) as well as cell intrinsic negative regulatory (costimulatory)pathways (PD-1, B7-H3, B7-H4, etc.).

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8+ T-cells,increased proliferation, increased antigen responsiveness (e.g., viral,pathogen, or tumor clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%enhancement. The manner of measuring this enhancement is known to one ofordinary skill in the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiveness to antigenic stimulation. In aparticular embodiment, a T-cell dysfunctional disorder is a disorderthat is specifically associated with inappropriate increased signalingthrough PD-1. In another embodiment, a T-cell dysfunctional disorder isone in which T-cells are anergic or have decreased ability to secretecytokines, proliferate, or execute cytolytic activity. In a specificaspect, the decreased responsiveness results in ineffective control of apathogen or tumor expressing an immunogen. Examples of T celldysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

“Immunogenicity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse. Examples of enhancing tumor immunogenicity include treatmentwith a PD-1 axis binding antagonist and a taxane.

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

As used herein, “reducing or inhibiting cancer relapse” means to reduceor inhibit tumor or cancer relapse or tumor or cancer progression. Asdisclosed herein, cancer relapse and/or cancer progression include,without limitation, cancer metastasis.

As used herein, “complete response” or “CR” refers to disappearance ofall target lesions.

As used herein, “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD.

As used herein, “stable disease” or “SD” refers to neither sufficientshrinkage of target lesions to qualify for PR, nor sufficient increaseto qualify for PD, taking as reference the smallest SLD since thetreatment started.

As used herein, “progressive disease” or “PD” refers to at least a 20%increase in the SLD of target lesions, taking as reference the smallestSLD recorded since the treatment started or the presence of one or morenew lesions.

As used herein, “progression free survival” (PFS) refers to the lengthof time during and after treatment during which the disease beingtreated (e.g., cancer) does not get worse. Progression-free survival mayinclude the amount of time patients have experienced a complete responseor a partial response, as well as the amount of time patients haveexperienced stable disease.

As used herein, “overall response rate” or “objective response rate”(ORR) refers to the sum of complete response (CR) rate and partialresponse (PR) rate.

As used herein, “overall survival” (OS) refers to the percentage ofindividuals in a group who are likely to be alive after a particularduration of time.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile. “Pharmaceuticallyacceptable” excipients (vehicles, additives) are those which canreasonably be administered to a subject mammal to provide an effectivedose of the active ingredient employed.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with cancer are mitigated or eliminated,including, but are not limited to, reducing the proliferation of (ordestroying) cancerous cells, decreasing symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, and/or prolonging survival of individuals.

As used herein, “delaying progression” of a disease means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

An “effective amount” is at least the minimum amount required to effecta measurable improvement or prevention of a particular disorder. Aneffective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe agent to elicit a desired response in the individual. An effectiveamount is also one in which any toxic or detrimental effects of thetreatment are outweighed by the therapeutically beneficial effects. Forprophylactic use, beneficial or desired results include results such aseliminating or reducing the risk, lessening the severity, or delayingthe onset of the disease, including biochemical, histological and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease.For therapeutic use, beneficial or desired results include clinicalresults such as decreasing one or more symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, and enhancing effect of another medication such as viatargeting, delaying the progression of the disease, and/or prolongingsurvival. In the case of a cancer or a tumor, an effective amount of thedrug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

A “disorder” is any condition that would benefit from treatmentincluding, but not limited to, chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer. In one embodiment, the cell proliferative disorder is a tumor.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder,” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. The term “breast cancer” includes, but is notlimited to, HER2+ breast cancer and triple-negative breast cancer(TNBC), which is a form of breast cancer in which the cancer cells arenegative for estrogen receptors (ER−), progesterone receptors (PR−), andHER2 (HER2−), and which may be locally advanced, unresectable, and/ormetastatic (e.g., metastatic triple-negative breast cancer (mTNBC)). Themethods described herein are suitable for treatment of various stages ofcancer, including cancers that are locally advanced and/or metastatic.In cancer staging, locally advanced is generally defined as cancer thathas spread from a localized area to nearby tissues and/or lymph nodes.In the Roman numeral staging system, locally advanced usually isclassified in Stage II or III. Cancer which is metastatic is a stagewhere the cancer spreads throughout the body to distant tissues andorgans (stage IV).

The term “cytotoxic agent” as used herein refers to any agent that isdetrimental to cells (e.g., causes cell death, inhibits proliferation,or otherwise hinders a cellular function). Cytotoxic agents include, butare not limited to, radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu);chemotherapeutic agents; growth inhibitory agents; enzymes and fragmentsthereof such as nucleolytic enzymes; and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Exemplarycytotoxic agents can be selected from anti-microtubule agents, platinumcoordination complexes, alkylating agents, antibiotic agents,topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine kinase angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A,inhibitors of fatty acid biosynthesis, cell cycle signalling inhibitors,HDAC inhibitors, proteasome inhibitors, and inhibitors of cancermetabolism. In one embodiment the cytotoxic agent is a platinum-basedchemotherapeutic agent. In one embodiment the cytotoxic agent is anantagonist of EGFR. In one embodiment the cytotoxic agent isN-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g.,erlotinib, TARCEVA™). In one embodiment the cytotoxic agent is a RAFinhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAFinhibitor. In one embodiment the RAF inhibitor is vemurafenib. In oneembodiment the cytotoxic agent is a PI3K inhibitor.

As used herein, the term “chemotherapeutic agent” includes compoundsuseful in the treatment of cancer, such as mTNBC. Examples ofchemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSIPharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram,epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG(geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant(FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer/Sugen), letrozole(FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate(VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU(5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth),Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafamib (SCH66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®,AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingtopotecan and irinotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);adrenocorticosteroids (including prednisone and prednisolone);cyproterone acetate; 5α-reductases including finasteride anddutasteride); vorinostat, romidepsin, panobinostat, valproic acid,mocetinostat dolastatin; aldesleukin, talc duocarmycin (including thesynthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlomaphazine, chlorophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin γ1I andcalicheamicin ω1 I (Angew Chem. Intl. Ed. Engl. 33:183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes;chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine;methotrexate; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Chemotherapeutic agents also include “platinum-based” chemotherapeuticagents, which comprise an organic compound which contains platinum as anintegral part of the molecule. Typically platinum-based chemotherapeuticagents are coordination complexes of platinum. Platinum-basedchemotherapeutic agents are sometimes called “platins” in the art.Examples of platinum-based chemotherapeutic agents include, but are notlimited to, carboplatin, cisplatin, and oxaliplatin.

Chemotherapeutic agents also include (i) anti-hormonal agents that actto regulate or inhibit hormone action on tumors such as anti-estrogensand selective estrogen receptor modulators (SERMs), including, forexample, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene,droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON® (toremifine citrate); (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate),AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR®(vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole;AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide and goserelin; buserelin, tripterelin,medroxyprogesterone acetate, diethylstilbestrol, premarin,fluoxymesterone, all transretionic acid, fenretinide, as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceuticallyacceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agents also include antibodies such as alemtuzumab(Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®,Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®,Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech),trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), andthe antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).Additional humanized monoclonal antibodies with therapeutic potential asagents in combination with the compounds of the invention include:apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine,cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab,cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab,felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin,ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab,motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab,numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab,pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab,ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695,Wyeth Research and Abbott Laboratories) which is a recombinantexclusively human-sequence, full-length IgG₁ λ antibody geneticallymodified to recognize interleukin-12 p40 protein.

Chemotherapeutic agents also include “EGFR inhibitors,” which refers tocompounds that bind to or otherwise interact directly with EGFR andprevent or reduce its signaling activity, and is alternatively referredto as an “EGFR antagonist.” Examples of such agents include antibodiesand small molecules that bind to EGFR. Examples of antibodies which bindto EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No.4,943,533) and variants thereof, such as chimerized 225 (C225 orCetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, e.g., WO96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targetedantibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat.No. 5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen);EMD 55900 (Stragliotto et al., Eur. J. Cancer 32A:636-640 (1996));EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR thatcompetes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); humanEGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known asE1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described inU.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanizedmAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Theanti-EGFR antibody may be conjugated with a cytotoxic agent, thusgenerating an immunoconjugate (see, e.g., EP659439A2, Merck PatentGmbH). EGFR antagonists include small molecules such as compoundsdescribed in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307,5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726,6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459,6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, aswell as the following PCT publications: WO98/14451, WO98/50038,WO99/09016, and WO99/24037. Particular small molecule EGFR antagonistsinclude OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSIPharmaceuticals); PD 183805 (CI 1033, 2-propenamide,N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-,dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®)4-(3′-Chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline,AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline,Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine,Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol);(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine);CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide);EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide)(Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 orN-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

Chemotherapeutic agents also include “tyrosine kinase inhibitors”including the EGFR-targeted drugs noted in the preceding paragraph;small molecule HER2 tyrosine kinase inhibitor such as TAK165 availablefrom Takeda; CP-724,714, an oral selective inhibitor of the ErbB2receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such asEKB-569 (available from Wyeth) which preferentially binds EGFR butinhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016;available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinaseinhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such ascanertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisenseagent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1signaling; non-HER-targeted tyrosine kinase inhibitors such as imatinibmesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targetedtyrosine kinase inhibitors such as sunitinib (SUTENT®, available fromPfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib(PTK787/ZK222584, available from Novartis/Schering AG); MAPKextracellular regulated kinase I inhibitor CI-1040 (available fromPharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules(e.g., those that bind to HER-encoding nucleic acid); quinoxalines (U.S.Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors suchas CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinibmesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone),rapamycin (sirolimus, RAPAMUNE®); or as described in any of thefollowing patent publications: U.S. Pat. No. 5,804,396; WO 1999/09016(American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983(Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (WarnerLambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferons,colchicine, metoprine, cyclosporine, amphotericin, metronidazole,alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide,asparaginase, BCG live, bevacuzimab, bexarotene, cladribine,clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa,elotinib, filgrastim, histrelin acetate, ibritumomab, interferonalfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna,methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin,palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pemetrexed disodium, plicamycin, porfimer sodium, quinacrine,rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene,tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, andpharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone, hydrocortisoneacetate, cortisone acetate, tixocortol pivalate, triamcinoloneacetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide,desonide, fluocinonide, fluocinolone acetonide, betamethasone,betamethasone sodium phosphate, dexamethasone, dexamethasone sodiumphosphate, fluocortolone, hydrocortisone-17-butyrate,hydrocortisone-17-valerate, aclometasone dipropionate, betamethasonevalerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate and fluprednidene acetate; immuneselective anti-inflammatory peptides (ImSAIDs) such asphenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG)(IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such asazathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts,hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumornecrosis factor alpha (TNFa) blockers such as etanercept (Enbrel),infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia),golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra(Kineret), T cell costimulation blockers such as abatacept (Orencia),Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®);Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha(IFN) blockers such as rontalizumab; Beta 7 integrin blockers such asrhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secretedhomotrimeric LTa3 and membrane bound heterotrimer LTa1/β2 blockers suchas Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu); miscellaneous investigational agents such asthioplatin, PS-341, phenylbutyrate, ET-18-OCH₃, or farnesyl transferaseinhibitors (L-739749, L-744832); polyphenols such as quercetin,resveratrol, piceatannol, epigallocatechine gallate, theaflavins,flavanols, procyanidins, betulinic acid and derivatives thereof;autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; acetylcamptothecin, scopolectin, and 9-am inocamptothecin);podophyllotoxin; tegafur (UFTORAL®)); bexarotene (TARGRETIN®));bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R);vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g.,celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; farnesyltransferaseinhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone; and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents also include non-steroidal anti-inflammatorydrugs with analgesic, antipyretic and anti-inflammatory effects. NSAIDsinclude non-selective inhibitors of the enzyme cyclooxygenase. Specificexamples of NSAIDs include aspirin, propionic acid derivatives such asibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen,acetic acid derivatives such as indomethacin, sulindac, etodolac,diclofenac, enolic acid derivatives such as piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivativessuch as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamicacid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib,parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicatedfor the symptomatic relief of conditions such as rheumatoid arthritis,osteoarthritis, inflammatory arthropathies, ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.In one embodiment, a growth inhibitory agent is growth inhibitoryantibody that prevents or reduces proliferation of a cell expressing anantigen to which the antibody binds. In another embodiment, the growthinhibitory agent may be one which significantly reduces the percentageof cells in S phase. Examples of growth inhibitory agents include agentsthat block cell cycle progression (at a place other than S phase), suchas agents that induce G1 arrest and M-phase arrest. Classical M-phaseblockers include the vincas (vincristine and vinblastine), taxanes, andtopoisomerase II inhibitors such as doxorubicin, epirubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in Mendelsohn and Israel, eds., The Molecular Basis of Cancer,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W.B. Saunders, Philadelphia,1995), e.g., p. 13.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone-time administration and typical dosages range from 10 to 200 units(Grays) per day.

An “individual” or a “subject” for purposes of treatment refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, horses, cats,cows, etc. Preferably, the mammal is human. A individual or subject maybe a patient.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. An isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, an isolatedantibody will be prepared by at least one purification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “constant domain” refers to the portion of an immunoglobulinmolecule having a more conserved amino acid sequence relative to theother portion of the immunoglobulin, the variable domain, which containsthe antigen binding site. The constant domain contains the C_(H)1,C_(H)2 and C_(H)3 domains (collectively, CH) of the heavy chain and theCHL (or CL) domain of the light chain.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “V_(H).” Thevariable domain of the light chain may be referred to as “V_(L).” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any mammalianspecies can be assigned to one of two clearly distinct types, calledkappa (“K”) and lambda (“A”), based on the amino acid sequences of theirconstant domains.

The term IgG “isotype” or “subclass” as used herein is meant any of thesubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, γ, ϵ, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding region thereof. In someembodiments, the antibody fragment described herein is anantigen-binding fragment. Examples of antibody fragments include Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention may be made by avariety of techniques, including, for example, the hybridoma method(e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S. Pat. No.5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg etal., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994);Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger,Nature Biotechnol. 14: 826 (1996); and Lonberg et al., Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATTZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen(e.g., has a binding affinity (Kd) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ M and preferably no more than about1×10⁻⁹ M) but has a binding affinity for a homologue of the antigen froma second nonhuman mammalian species which is at least about 50 fold, orat least about 500 fold, or at least about 1000 fold, weaker than itsbinding affinity for the human antigen. The species-dependent antibodycan be any of the various types of antibodies as defined above, butpreferably is a humanized or human antibody.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

As used herein, the term “binds”, “specifically binds to” or is“specific for” refers to measurable and reproducible interactions suchas binding between a target and an antibody, which is determinative ofthe presence of the target in the presence of a heterogeneous populationof molecules including biological molecules. For example, an antibodythat binds to or specifically binds to a target (which can be anepitope) is an antibody that binds this target with greater affinity,avidity, more readily, and/or with greater duration than it binds toother targets. In one embodiment, the extent of binding of an antibodyto an unrelated target is less than about 10% of the binding of theantibody to the target as measured, e.g., by a radioimmunoassay (RIA).In certain embodiments, an antibody that specifically binds to a targethas a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or≤0.1 nM. In certain embodiments, an antibody specifically binds to anepitope on a protein that is conserved among the protein from differentspecies. In another embodiment, specific binding can include, but doesnot require exclusive binding.

II. PD-1 Axis Binding Antagonists

Provided herein are methods for treating or delaying progression oflocally advanced or metastatic breast cancer (e.g., mTNBC) in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and a taxane. Also providedherein are methods of enhancing immune function in an individual havinglocally advanced or metastatic breast cancer (e.g., mTNBC) comprisingadministering to the individual an effective amount of a PD-1 axisbinding antagonist and an taxane. For example, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist. PD-1 (programmed death 1) isalso referred to in the art as “programmed cell death 1,” “PDCD1,”“CD279,” and “SLEB2.” An exemplary human PD-1 is shown inUniProtKB/Swiss-Prot Accession No. 015116. PD-L1 (programmed deathligand 1) is also referred to in the art as “programmed cell death 1ligand 1,” “PDCD1LG1,” “CD274,” “B7-H,” and “PDL1.” An exemplary humanPD-L1 is shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1. PD-L2(programmed death ligand 2) is also referred to in the art as“programmed cell death 1 ligand 2,” “PDCD1 LG2,” “CD273,” “B7-DC,”“Btdc,” and “PDL2.” An exemplary human PD-L2 is shown inUniProtKB/Swiss-Prot Accession No. Q9BQ51. In some embodiments, PD-1,PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect the PD-1 ligand binding partners are PD-L1 and/or PD-L2.In another embodiment, a PD-L1 binding antagonist is a molecule thatinhibits the binding of PD-L1 to its binding partners. In a specificaspect, PD-L1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PD-L2 binding antagonist is a molecule that inhibits thebinding of PD-L2 to its binding partners. In a specific aspect, a PD-L2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1antibody (e.g., a human antibody, a humanized antibody, or a chimericantibody). In some embodiments, the anti-PD-1 antibody is selected fromthe group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab),CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, andBGB-108. In some embodiments, the PD-1 binding antagonist is animmunoadhesin (e.g., an immunoadhesin comprising an extracellular orPD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g.,an Fc region of an immunoglobulin sequence). In some embodiments, thePD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1binding antagonist is anti-PD-L1 antibody. In some embodiments, theanti-PD-L1 antibody is selected from the group consisting of MPDL3280A,YW243.55.S70, MDX-1105, MED14736 (durvalumab), and MSB0010718C(avelumab). Antibody YW243.55.S70 is an anti-PD-L1 described in WO2010/077634. MDX-1105, also known as BMS-936559, is an anti-PD-L1antibody described in WO2007/005874. MED14736 is an anti-PD-L1monoclonal antibody described in WO2011/066389 and US2013/034559.MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab,is an anti-PD-1 antibody described in WO2006/121168. MK-3475, also knownas lambrolizumab, is an anti-PD-1 antibody described in WO2009/114335.CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, isa PD-L2-Fc fusion soluble receptor described in WO2010/027827 andWO2011/066342.

In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1antibody. In some embodiments, the anti-PD-L1 antibody is capable ofinhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1.In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody.In some embodiments, the anti-PD-L1 antibody is an antibody fragmentselected from the group consisting of Fab, Fab′-SH, Fv, scFv, and(Fab′)₂ fragments. In some embodiments, the anti-PD-L1 antibody is ahumanized antibody. In some embodiments, the anti-PD-L1 antibody is ahuman antibody.

Examples of anti-PD-L1 antibodies useful for the methods of thisinvention, and methods for making thereof are described in PCT patentapplication WO 2010/077634, WO 2007/005874, WO 2011/066389, and US2013/034559, which are incorporated herein by reference. The anti-PD-L1antibodies useful in this invention, including compositions containingsuch antibodies, may be used in combination with a taxane to treatcancer.

Anti-PD-1 Antibodies

In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternativenames for “MDX-1106” include MDX-1106-04, ONO-4538, BMS-936558, ornivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CASRegistry Number: 946414-94-4). In a still further embodiment, providedis an isolated anti-PD-1 antibody comprising a heavy chain variableregion comprising the heavy chain variable region amino acid sequencefrom SEQ ID NO:1 and/or a light chain variable region comprising thelight chain variable region amino acid sequence from SEQ ID NO:2. In astill further embodiment, provided is an isolated anti-PD-1 antibodycomprising a heavy chain and/or a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:1), and

(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 2) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFlFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Anti-PD-L1 Antibodies

In some embodiments, the antibody in the formulation comprises at leastone tryptophan (e.g., at least two, at least three, or at least four) inthe heavy and/or light chain sequence. In some embodiments, amino acidtryptophan is in the HVR regions, framework regions and/or constantregions of the antibody. In some embodiments, the antibody comprises twoor three tryptophan residues in the HVR regions. In some embodiments,the antibody in the formulation is an anti-PD-L1 antibody. PD-L1(programmed death ligand 1), also known as PDL1, B7-H1, B7-4, CD274, andB7-H, is a transmembrane protein, and its interaction with PD-1 inhibitsT-cell activation and cytokine production. In some embodiments, theanti-PD-L1 antibody described herein binds to human PD-L1. Examples ofanti-PD-L1 antibodies that can be used in the methods described hereinare described in PCT patent application WO 2010/077634 A1 and U.S. Pat.No. 8,217,149, which are incorporated herein by reference in theirentirety.

In some embodiments, the anti-PD-L1 antibody is capable of inhibitingbinding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In someembodiments, the anti-PD-L1 antibody is a monoclonal antibody. In someembodiments, the anti-PD-L1 antibody is an antibody fragment selectedfrom the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab′)₂fragments. In some embodiments, the anti-PD-L1 antibody is a humanizedantibody. In some embodiments, the anti-PD-L1 antibody is a humanantibody.

Anti-PD-L1 antibodies described in WO 2010/077634 A1 and U.S. Pat. No.8,217,149 may be used in the methods described herein. In someembodiments, the anti-PD-L1 antibody comprises a heavy chain variableregion sequence of SEQ ID NO:3 and/or a light chain variable regionsequence of SEQ ID NO:4. In a still further embodiment, provided is anisolated anti-PD-L1 antibody comprising a heavy chain variable regionand/or a light chain variable region sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:3), and

(b) the light chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYLYHPATFGQGTKVEIKR.

In one embodiment, the anti-PD-L1 antibody comprises a heavy chainvariable region comprising an HVR-H1, HVR-H2 and HVR-H3 sequence,wherein:

(a) the HVR-H1 sequence is (SEQ ID NO: 5) GFTFSX₁SWIH;(b) the HVR-H2 sequence is (SEQ ID NO: 6) AWIX₂PYGGSX₃YYADSVKG;(c) the HVR-H3 sequence is (SEQ ID NO: 7) RHWPGGFDY;

further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S. In onespecific aspect, X₁ is D; X₂ is S and X₃ is T.

In another aspect, the polypeptide further comprises variable regionheavy chain framework sequences juxtaposed between the HVRs according tothe formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the framework sequences are VHsubgroup III consensus framework. In a still further aspect, at leastone of the framework sequences is the following:

HC-FR1 is (SEQ ID NO: 8) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 is(SEQ ID NO: 9) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 10)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 is (SEQ ID NO: 11) WGQGTLVTVSA.

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain comprising an HVR-L1, HVR-L2and HVR-L3, wherein:

(a) the HVR-L1 sequence is (SEQ ID NO: 12) RASQX₄X₅X₆TX₇X₈A;(b) the HVR-L2 sequence is (SEQ ID NO: 13) SASX₉LX₁₀S;(c) the HVR-L3 sequence is (SEQ ID NO: 14) QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;wherein: X₄ is D or V; X₅ is V or I; X₆ is S or N; X₇ is A or F; X₈ is Vor L; X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G, F, or S; X₁₂ is L, Y, For W; X₁₃ is Y, N, A, T, G, F or I; X₁₄ is H, V, P, Tor I; X₁₅ is A, W,R, P or T. In a still further aspect, X₄ is D; X₅ is V; X₆ is S; X₇ isA; X₈ is V; X₉ is F; X₁₀ is Y; X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H;X₁₅ is A.

In a still further aspect, the light chain further comprises variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the frameworksequences are VL kappa I consensus framework. In a still further aspect,at least one of the framework sequence is the following:

LC-FR1 is (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITC LC-FR2 is(SEQ ID NO: 16) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 17)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 18) FGQGTKVEIKR.

In another embodiment, provided is an isolated anti-PD-L1 antibody orantigen binding fragment comprising a heavy chain and a light chainvariable region sequence, wherein:

(a) the heavy chain comprises an HVR-H1, HVR-H2 and HVR-H3, whereinfurther:

-   -   (i) the HVR-H1 sequence is GFTFSX₁SWIH; (SEQ ID NO:5)    -   (ii) the HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG (SEQ ID NO:6)    -   (iii) the HVR-H3 sequence is RHWPGGFDY, and (SEQ ID NO:7)

(b) the light chain comprises an HVR-L1, HVR-L2 and HVR-L3, whereinfurther:

(i) the HVR-L1 sequence is (SEQ ID NO: 12) RASQX₄X₅X₆TX₇X₈A(ii) the HVR-L2 sequence is (SEQ ID NO: 13) SASX₉LX₁₀S; and(iii) the HVR-L3 sequence is (SEQ ID NO: 14) QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S; X₄ is D or V; X₅ is Vor I; X₆ is S or N; X₇ is A or F; X₈ is V or L; X₉ is F or T; X₁₀ is Yor A; X₁₁ is Y, G, F, or S; X₁₂ is L, Y, F or W; X₁₃ is Y, N, A, T, G, For I; X₁₄ is H, V, P, T or I; X₁₅ is A, W, R, P or T. In a specificaspect, X₁ is D; X₂ is S and X₃ is T. In another aspect, X₄ is D; X₅ isV; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀ is Y; X₁₁ is Y; X₁₂ is L; X₁₃is Y; X₁₄ is H; X₁₅ is A. In yet another aspect, X₁ is D; X₂ is Sand X₃is T, X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀ is Y;X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H and X₁₅ is A.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences are set forthas SEQ ID NOs:8, 9, 10 and 11. In a still further aspect, the lightchain framework sequences are derived from a Kabat kappa I, II, II or IVsubgroup sequence. In a still further aspect, the light chain frameworksequences are VL kappa I consensus framework. In a still further aspect,one or more of the light chain framework sequences are set forth as SEQID NOs:15, 16, 17 and 18.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

-   -   (a) the heavy chain further comprises an HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and        RHWPGGFDY (SEQ ID NO:21), respectively, or    -   (b) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:22), SASFLYS (SEQ ID NO:23) and QQYLYHPAT        (SEQ ID NO:24), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another aspect, the heavy chain variable region comprises one or moreframework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup I, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences are set forth as SEQ IDNOs:8, 9, 10 and 11. In a still further aspect, the light chainframework sequences are derived from a Kabat kappa I, II, II or IVsubgroup sequence. In a still further aspect, the light chain frameworksequences are VL kappa I consensus framework. In a still further aspect,one or more of the light chain framework sequences are set forth as SEQID NOs:15, 16, 17 and 18.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In another further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:25), and/or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYLYHPATFGQGTKVEIKR.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9,10 and WGQGTLVTVSS (SEQ ID NO:27).

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences are set forth as SEQ ID NOs:15, 16, 17 and 18.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences is thefollowing:

HC-FR1 (SEQ ID NO: 29) EVQLVESGGGLVQPGGSLRLSCAASGFTFS HC-FR2(SEQ ID NO: 30) WVRQAPGKGLEWVA HC-FR3 (SEQ ID NO: 10)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 27) WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 16)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 17) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 28) FGQGTKVEIK.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

-   -   (c) the heavy chain further comprises an HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and        RHWPGGFDY (SEQ ID NO:21), respectively, and/or    -   (d) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:22), SASFLYS (SEQ ID NO:23) and QQYLYHPAT        (SEQ ID NO:24), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another aspect, the heavy chain variable region comprises one or moreframework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup I, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences are set forth as SEQ IDNOs:8, 9, 10 and WGQGTLVTVSSASTK (SEQ ID NO:31).

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences are set forth as SEQ ID NOs:15, 16, 17 and 18.In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region. In a still furtherembodiment, provided is an isolated anti-PD-L1 antibody comprising aheavy chain and a light chain variable region sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:26), or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYLYHPATFGQGTKVEIKR.

In some embodiments, provided is an isolated anti-PD-L1 antibodycomprising a heavy chain and a light chain variable region sequence,wherein the light chain variable region sequence has at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:4. In some embodiments,provided is an isolated anti-PD-L1 antibody comprising a heavy chain anda light chain variable region sequence, wherein the heavy chain variableregion sequence has at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to the amino acid sequenceof SEQ ID NO:26. In some embodiments, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein the light chain variable region sequence has at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:4 and theheavy chain variable region sequence has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO:26. In some embodiments, one, two,three, four or five amino acid residues at the N-terminal of the heavyand/or light chain may be deleted, substituted or modified.

In a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:32), and/or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 33) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In some embodiments, provided is an isolated anti-PD-L1 antibodycomprising a heavy chain and a light chain sequence, wherein the lightchain sequence has at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:33. In some embodiments, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain sequence, whereinthe heavy chain sequence has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:32. In some embodiments, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain sequence, whereinthe light chain sequence has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:33 and the heavy chain sequence has at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO:32.

In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Removal of glycosylation sites form anantibody is conveniently accomplished by altering the amino acidsequence such that one of the above-described tripeptide sequences (forN-linked glycosylation sites) is removed. The alteration may be made bysubstitution of an asparagine, serine or threonine residue within theglycosylation site another amino acid residue (e.g., glycine, alanine ora conservative substitution).

In any of the embodiments herein, the isolated anti-PD-L1 antibody canbind to a human PD-L1, for example a human PD-L1 as shown inUniProtKB/Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof.

In a still further embodiment, provided is an isolated nucleic acidencoding any of the antibodies described herein. In some embodiments,the nucleic acid further comprises a vector suitable for expression ofthe nucleic acid encoding any of the previously described anti-PD-L1antibodies. In a still further specific aspect, the vector is in a hostcell suitable for expression of the nucleic acid. In a still furtherspecific aspect, the host cell is a eukaryotic cell or a prokaryoticcell. In a still further specific aspect, the eukaryotic cell is amammalian cell, such as Chinese hamster ovary (CHO) cell.

The antibody or antigen binding fragment thereof, may be made usingmethods known in the art, for example, by a process comprising culturinga host cell containing nucleic acid encoding any of the previouslydescribed anti-PD-L1 antibodies or antigen-binding fragment in a formsuitable for expression, under conditions suitable to produce suchantibody or fragment, and recovering the antibody or fragment.

III. Antibody Preparation

The antibodies described herein are prepared using techniques availablein the art for generating antibodies, exemplary methods of which aredescribed in more detail in the following sections.

The antibody is directed against an antigen of interest (e.g., PD-L1(such as a human PD-L1), PD1 (such as human PD-L1), PD-L2 (such as humanPD-L2), etc.). Preferably, the antigen is a biologically importantpolypeptide and administration of the antibody to a mammal sufferingfrom a disorder can result in a therapeutic benefit in that mammal.

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of 1 μM, 150 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01nM, or 0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g.,from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 μM or 26 μM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest. The Fab of interest isthen incubated overnight; however, the incubation may continue for alonger period (e.g., about 65 hours) to ensure that equilibrium isreached. Thereafter, the mixtures are transferred to the capture platefor incubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips atapproximately 10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml(approximately 0.2 μM) before injection at a flow rate of 5 μl/minute toachieve approximately 10 response units (RU) of coupled protein.Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on) See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonanceassay above, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g.,the extracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.,cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Certain Antibody-Based Methods

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (i.p.) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal antibodies of the invention can be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), andfurther described, for example, in Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) regardinghuman-human hybridomas. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 regarding production of monoclonalhuman natural IgM antibodies from hybridoma cell lines. Human hybridomatechnology (Trioma technology) is described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

For various other hybridoma techniques, see, for example, U.S. PatentPublication Nos. 2006/258841; 2006/183887 (fully human antibodies),2006/059575; 2005/287149; 2005/100546; and 2005/026229; and U.S. Pat.Nos. 7,078,492 and 7,153,507. An exemplary protocol for producingmonoclonal antibodies using the hybridoma method is described asfollows. In one embodiment, a mouse or other appropriate host animal,such as a hamster, is immunized to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies are raised in animals bymultiple subcutaneous (SC) or intraperitoneal (IP) injections of apolypeptide of the invention or a fragment thereof, and an adjuvant,such as monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM)(Ribi Immunochem. Research, Inc., Hamilton, Mont.). A polypeptide of theinvention (e.g., antigen) or a fragment thereof may be prepared usingmethods well known in the art, such as recombinant methods, some ofwhich are further described herein. Serum from immunized animals isassayed for anti-antigen antibodies, and booster immunizations areoptionally administered. Lymphocytes from animals producing anti-antigenantibodies are isolated. Alternatively, lymphocytes may be immunized invitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies that bind to an antibody of theinvention. The binding specificity of monoclonal antibodies produced byhybridoma cells may be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA). The binding affinity of the monoclonalantibody can be determined, for example, by Scatchard analysis. See,e.g., Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, hybridomacells may be grown in vivo as ascites tumors in an animal. Monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography. One procedure for isolation of proteins fromhybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method includes using minimal salts, such as lyotropicsalts, in the binding process and preferably also using small amounts oforganic solvents in the elution process.

(iii) Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Additional methods are reviewed,e.g., in Hoogenboom et al., in Methods in Molecular Biology 178:1-37(O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and furtherdescribed, e.g., in McCafferty et al., Nature 348:552-554; Clackson etal., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J.Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self-antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

(iv) Chimeric, Humanized and Human Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), for example, torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993));

framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285(1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature(somatically mutated) framework regions or human germline frameworkregions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633(2008)); and framework regions derived from screening FR libraries (see,e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok etal., J. Biol. Chem. 271:22611-22618 (1996)).

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, e.g., by combiningwith a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

(v) Antibody Fragments

Antibody fragments may be generated by traditional means, such asenzymatic digestion, or by recombinant techniques. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors. For areview of certain antibody fragments, see Hudson et al. (2003) Nat. Med.9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)2 fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)2 fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)2 fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See, for example, WO 93/16185; U.S. Pat. Nos.5,571,894; and 5,587,458. Fv and scFv are the only species with intactcombining sites that are devoid of constant regions; thus, they may besuitable for reduced nonspecific binding during in vivo use. scFv fusionproteins may be constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an scFv. See AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment may also be a“linear antibody,” e.g., as described in U.S. Pat. No. 5,641,870, forexample. Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two differentepitopes (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Bispecific antibodies can be prepared asfull-length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, e.g., Millsteinet al., Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

One approach known in the art for making bispecific antibodies is the“knobs-into-holes” or “protuberance-into-cavity” approach (see, e.g.,U.S. Pat. No. 5,731,168). In this approach, two immunoglobulinpolypeptides (e.g., heavy chain polypeptides) each comprise aninterface. An interface of one immunoglobulin polypeptide interacts witha corresponding interface on the other immunoglobulin polypeptide,thereby allowing the two immunoglobulin polypeptides to associate. Theseinterfaces may be engineered such that a “knob” or “protuberance” (theseterms may be used interchangeably herein) located in the interface ofone immunoglobulin polypeptide corresponds with a “hole” or “cavity”(these terms may be used interchangeably herein) located in theinterface of the other immunoglobulin polypeptide. In some embodiments,the hole is of identical or similar size to the knob and suitablypositioned such that when the two interfaces interact, the knob of oneinterface is positionable in the corresponding hole of the otherinterface. Without wishing to be bound to theory, this is thought tostabilize the heteromultimer and favor formation of the heteromultimerover other species, for example homomultimers. In some embodiments, thisapproach may be used to promote the heteromultimerization of twodifferent immunoglobulin polypeptides, creating a bispecific antibodycomprising two immunoglobulin polypeptides with binding specificitiesfor different epitopes.

In some embodiments, a knob may be constructed by replacing a smallamino acid side chain with a larger side chain. In some embodiments, ahole may be constructed by replacing a large amino acid side chain witha smaller side chain. Knobs or holes may exist in the originalinterface, or they may be introduced synthetically. For example, knobsor holes may be introduced synthetically by altering the nucleic acidsequence encoding the interface to replace at least one “original” aminoacid residue with at least one “import” amino acid residue. Methods foraltering nucleic acid sequences may include standard molecular biologytechniques well known in the art. The side chain volumes of variousamino acid residues are shown in the following table. In someembodiments, original residues have a small side chain volume (e.g.,alanine, asparagine, aspartic acid, glycine, serine, threonine, orvaline), and import residues for forming a knob are naturally occurringamino acids and may include arginine, phenylalanine, tyrosine, andtryptophan. In some embodiments, original residues have a large sidechain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan),and import residues for forming a hole are naturally occurring aminoacids and may include alanine, serine, threonine, and valine.

TABLE 1 Properties of amino acid residues One-letter Mass^(a) Volume^(b)Accessible surface Amino acid abbreviation (daltons) (Å³) area^(c) (Å²)Alanine (Ala) A 71.08 88.6 115 Arginine (Arg) R 156.20 173.4 225Asparagine (Asn) N 114.11 117.7 160 Aspartic Acid (Asp) D 115.09 111.1150 Cysteine (Cys) C 103.14 108.5 135 Glutamine (Gln) Q 128.14 143.9 180Glutamic Acid (Glu) E 129.12 138.4 190 Glycine (Gly) G 57.06 60.1 75Histidine (His) H 137.15 153.2 195 Isoleucine (Ile) I 113.17 166.7 175Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K 128.18 168.6 200Methionine (Met) M 131.21 162.9 185 Phenylalanine (Phe) F 147.18 189.9210 Proline (Pro) P 97.12 122.7 145 Serine (Ser) S 87.08 89.0 115Threonine (Thr) T 101.11 116.1 140 Tryptophan (Trp) W 186.21 227.8 255Tyrosine (Tyr) Y 163.18 193.6 230 Valine (Val) V 99.14 140.0 155^(a)Molecular weight of amino acid minus that of water. Values fromHandbook of Chemistry and Physics, 43^(rd) ed. Cleveland, ChemicalRubber Publishing Co., 1961. ^(b)Values from A.A. Zamyatnin, Prog.Biophys. Mol. Biol. 24: 107-123, 1972. ^(c)Values from C. Chothia, J.Mol. Biol. 105: 1-14, 1975. The accessible surface area is defined inFigures 6-20 of this reference.

In some embodiments, original residues for forming a knob or hole areidentified based on the three-dimensional structure of theheteromultimer. Techniques known in the art for obtaining athree-dimensional structure may include X-ray crystallography and NMR.In some embodiments, the interface is the CH3 domain of animmunoglobulin constant domain. In these embodiments, the CH3/CH3interface of human IgG₁ involves sixteen residues on each domain locatedon four anti-parallel β-strands. Without wishing to be bound to theory,mutated residues are preferably located on the two central anti-parallelβ-strands to minimize the risk that knobs can be accommodated by thesurrounding solvent, rather than the compensatory holes in the partnerCH3 domain. In some embodiments, the mutations forming correspondingknobs and holes in two immunoglobulin polypeptides correspond to one ormore pairs provided in the following table.

TABLE 2 Exemplary sets of corresponding knob-and hole-forming mutationsCH3 of first immunoglobulin CH3 of second immunoglobulin T366Y Y407TT366W Y407A F405A T394W Y407T T366Y T366Y:F405A T394W:Y407T T366W:F405WT394S:Y407A F405W:Y407A T366W:T394S F405W T394S Mutations are denoted bythe original residue, followed by the position using the Kabat numberingsystem, and then the import residue (all residues are given insingle-letter amino acid code). Multiple mutations are separated by acolon.

In some embodiments, an immunoglobulin polypeptide comprises a CH3domain comprising one or more amino acid substitutions listed in Table 2above. In some embodiments, a bispecific antibody comprises a firstimmunoglobulin polypeptide comprising a CH3 domain comprising one ormore amino acid substitutions listed in the left column of Table 2, anda second immunoglobulin polypeptide comprising a CH3 domain comprisingone or more corresponding amino acid substitutions listed in the rightcolumn of Table 2.

Following mutation of the DNA as discussed above, polynucleotidesencoding modified immunoglobulin polypeptides with one or morecorresponding knob- or hole-forming mutations may be expressed andpurified using standard recombinant techniques and cell systems known inthe art. See, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333;7,642,228; 7,695,936; 8,216,805; U.S. Pub. No. 2013/0089553; and Spiesset al., Nature Biotechnology 31: 753-758, 2013. Modified immunoglobulinpolypeptides may be produced using prokaryotic host cells, such as E.coli, or eukaryotic host cells, such as CHO cells. Correspondingknob-and-hole-bearing immunoglobulin polypeptides may be expressed inhost cells in co-culture and purified together as a heteromultimer, orthey may be expressed in single cultures, separately purified, andassembled in vitro. In some embodiments, two strains of bacterial hostcells (one expressing an immunoglobulin polypeptide with a knob, and theother expressing an immunoglobulin polypeptide with a hole) areco-cultured using standard bacterial culturing techniques known in theart. In some embodiments, the two strains may be mixed in a specificratio, e.g., so as to achieve equal expression levels in culture. Insome embodiments, the two strains may be mixed in a 50:50, 60:40, or70:30 ratio. After polypeptide expression, the cells may be lysedtogether, and protein may be extracted. Standard techniques known in theart that allow for measuring the abundance of homo-multimeric vs.hetero-multimeric species may include size exclusion chromatography. Insome embodiments, each modified immunoglobulin polypeptide is expressedseparately using standard recombinant techniques, and they may beassembled together in vitro. Assembly may be achieved, for example, bypurifying each modified immunoglobulin polypeptide, mixing andincubating them together in equal mass, reducing disulfides (e.g., bytreating with dithiothreitol), concentrating, and reoxidizing thepolypeptides. Formed bispecific antibodies may be purified usingstandard techniques including cation-exchange chromatography andmeasured using standard techniques including size exclusionchromatography. For a more detailed description of these methods, seeSpeiss et al., Nat. Biotechnol. 31:753-8, 2013. In some embodiments,modified immunoglobulin polypeptides may be expressed separately in CHOcells and assembled in vitro using the methods described above.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is typical to have thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. One interface comprises at least a part of the CH 3 domain ofan antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al, J. Immunol, 152:5368 (1994).

Another technique for making bispecific antibody fragments is the“bispecific T cell engager” or BiTE® approach (see, e.g., WO2004/106381,WO2005/061547, WO2007/042261, and WO2008/119567). This approach utilizestwo antibody variable domains arranged on a single polypeptide. Forexample, a single polypeptide chain includes two single chain Fv (scFv)fragments, each having a variable heavy chain (V_(H)) and a variablelight chain (V_(L)) domain separated by a polypeptide linker of a lengthsufficient to allow intramolecular association between the two domains.This single polypeptide further includes a polypeptide spacer sequencebetween the two scFv fragments. Each scFv recognizes a differentepitope, and these epitopes may be specific for different cell types,such that cells of two different cell types are brought into closeproximity or tethered when each scFv is engaged with its cognateepitope. One particular embodiment of this approach includes a scFvrecognizing a cell-surface antigen expressed by an immune cell, e.g., aCD3 polypeptide on a T cell, linked to another scFv that recognizes acell-surface antigen expressed by a target cell, such as a malignant ortumor cell.

As it is a single polypeptide, the bispecific T cell engager may beexpressed using any prokaryotic or eukaryotic cell expression systemknown in the art, e.g., a CHO cell line. However, specific purificationtechniques (see, e.g., EP1691833) may be necessary to separate monomericbispecific T cell engagers from other multimeric species, which may havebiological activities other than the intended activity of the monomer.In one exemplary purification scheme, a solution containing secretedpolypeptides is first subjected to a metal affinity chromatography, andpolypeptides are eluted with a gradient of imidazole concentrations.This eluate is further purified using anion exchange chromatography, andpolypeptides are eluted using with a gradient of sodium chlorideconcentrations. Finally, this eluate is subjected to size exclusionchromatography to separate monomers from multimeric species.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. See, e.g., Tuft et al. J.Immunol. 147: 60 (1991).

(vii) Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

(viii) Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

(ix) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 3 Exemplary Substitutions. Preferred Original Residue ExemplarySubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

c. acidic: Asp, Glu;

d. basic: His, Lys, Arg;

e. residues that influence chain orientation: Gly, Pro;

f. aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process (see, e.g.,Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs),with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001)). In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

(x) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided comprising an Fcregion wherein a carbohydrate structure attached to the Fc region hasreduced fucose or lacks fucose, which may improve ADCC function.Specifically, antibodies are contemplated herein that have reducedfucose relative to the amount of fucose on the same antibody produced ina wild-type CHO cell. That is, they are characterized by having a loweramount of fucose than they would otherwise have if produced by nativeCHO cells (e.g., a CHO cell that produce a native glycosylation pattern,such as, a CHO cell containing a native FUT8 gene). In certainembodiments, the antibody is one wherein less than about 50%, 40%, 30%,20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. Forexample, the amount of fucose in such an antibody may be from 1% to 80%,from 1% to 65%, from 5% to 65% or from 20% to 40%. In certainembodiments, the antibody is one wherein none of the N-linked glycansthereon comprise fucose, i.e., wherein the antibody is completelywithout fucose, or has no fucose or is afucosylated. The amount offucose is determined by calculating the average amount of fucose withinthe sugar chain at Asn297, relative to the sum of all glycostructuresattached to Asn 297 (e. g. complex, hybrid and high mannose structures)as measured by MALDI-TOF mass spectrometry, as described in WO2008/077546, for example. Asn297 refers to the asparagine residuelocated at about position 297 in the Fc region (EU numbering of Fcregion residues); however, Asn297 may also be located about ±3 aminoacids upstream or downstream of position 297, i.e., between positions294 and 300, due to minor sequence variations in antibodies. Suchfucosylation variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1; and WO 2004/056312 A1, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; US 2005/0123546, and Ferrara et al., Biotechnology andBioengineering, 93(5): 851-861 (2006). Antibody variants with at leastone galactose residue in the oligosaccharide attached to the Fc regionare also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, e.g., in WO 1997/30087;WO 1998/58964; and WO 1999/22764.

In certain embodiments, the antibody variants comprising an Fc regiondescribed herein are capable of binding to an FcγRIII. In certainembodiments, the antibody variants comprising an Fc region describedherein have ADCC activity in the presence of human effector cells orhave increased ADCC activity in the presence of human effector cellscompared to the otherwise same antibody comprising a human wild-typeIgG1Fc region.

(xi) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g., a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII,and FcγRIII. FcR expression on hematopoietic cells is summarized inTable 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492(1991). Non-limiting examples of in vitro assays to assess ADCC activityof a molecule of interest is described in U.S. Pat. No. 5,500,362 (see,e.g. Hellstrom et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986))and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052(2003); and Cragg et al, Blood 103:2738-2743 (2004)). FcRn binding andin vivo clearance/half life determinations can also be performed usingmethods known in the art (see, e.g., Petkova et al., Int'l. Immunol.18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues). In an exemplary embodiment, the antibodycomprising the following amino add substitutions in its Fc region:S298A, E333A, and K334A.

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.)). Those antibodies comprise an Fcregion with one or more substitutions therein which improve binding ofthe Fc region to FcRn. Such Fc variants include those with substitutionsat one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305,307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or434, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

(xii) Antibody Derivatives

The antibodies of the invention can be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. In certain embodiments, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

(xiii) Vectors, Host Cells, and Recombinant Methods

Antibodies may also be produced using recombinant methods. Forrecombinant production of an anti-antigen antibody, nucleic acidencoding the antibody is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or for expression. DNAencoding the antibody may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(a) Signal Sequence Component

An antibody of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (e.g., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process a native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, Ipp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

(b) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ, plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter.

(c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(d) Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(e) Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(g) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half-life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to theinvention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 255-268. Host cells are transformed with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

(h) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(xiv) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

(xv) Selecting Biologically Active Antibodies

Antibodies produced as described above may be subjected to one or more“biological activity” assays to select an antibody with beneficialproperties from a therapeutic perspective or selecting formulations andconditions that retain biological activity of the antibody. The antibodymay be tested for its ability to bind the antigen against which it wasraised. For example, methods known in the art (such as ELISA, WesternBlot, etc.) may be used.

For example, for an anti-PD-L1 antibody, the antigen binding propertiesof the antibody can be evaluated in an assay that detects the ability tobind to PD-L1. In some embodiments, the binding of the antibody may bedetermined by saturation binding; ELISA; and/or competition assays (e.g.RIA's), for example. Also, the antibody may be subjected to otherbiological activity assays, e.g., in order to evaluate its effectivenessas a therapeutic. Such assays are known in the art and depend on thetarget antigen and intended use for the antibody. For example, thebiological effects of PD-L1 blockade by the antibody can be assessed inCD8+ T cells, a lymphocytic choriomeningitis virus (LCMV) mouse modeland/or a syngeneic tumor model e.g., as described in U.S. Pat. No.8,217,149.

To screen for antibodies which bind to a particular epitope on theantigen of interest (e.g., those which block binding of the anti-PD-L1antibody of the example to PD-L1), a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270:1388-1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

IV. Pharmaceutical Compositions and Formulations

Also provided herein are pharmaceutical compositions and formulationscomprising a PD-1 axis binding antagonist and/or an antibody describedherein (such as an anti-PD-L1 antibody) and, optionally, apharmaceutically acceptable carrier. The invention also providespharmaceutical compositions and formulations comprising taxanes, e.g.,nab-paclitaxel (ABRAXANE®), paclitaxel, or docetaxel.

Pharmaceutical compositions and formulations as described herein can beprepared by mixing the active ingredients (e.g., a PD-1 axis bindingantagonist and/or a taxane) having the desired degree of purity with oneor more optional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The compositions and formulations herein may also contain more than oneactive ingredients as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. The formulationsto be used for in vivo administration are generally sterile. Sterilitymay be readily accomplished, e.g., by filtration through sterilefiltration membranes.

IV. Methods of Treatment

Provided herein are methods for treating or delaying progression oflocally advanced or metastatic breast cancer (e.g., mTNBC) in anindividual comprising administering to the individual an effectiveamount of a PD-1 axis binding antagonist and a taxane (e.g.,nab-paclitaxel (ABRAXANE®) or paclitaxel). In some embodiments, thetreatment results in a response in the individual after treatment. Insome embodiments, the response is a partial response. In someembodiments, the response is a complete response. In some embodiments,the treatment results in a sustained response (e.g., a sustained partialresponse or complete response) in the individual after cessation of thetreatment. The methods described herein may find use in treatingconditions where enhanced immunogenicity is desired such as increasingtumor immunogenicity for the treatment of cancer. Also provided hereinare methods of enhancing immune function in an individual having alocally advanced or metastatic breast cancer comprising administering tothe individual an effective amount of a PD-1 axis binding antagonist(e.g., MPDL3280A) and a taxane (e.g., nab-paclitaxel (ABRAXANE®)). Anyof the PD-1 axis binding antagonists and the taxanes known in the art ordescribed herein may be used in the methods.

In some instances, the methods provided herein include administration ofan effective amount of a PD-1 axis binding antagonist is selected fromthe group consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist, and a PD-L2 binding antagonist. In some instances, the PD-L1binding antagonist is an antibody, such as an antibody that is capableof inhibiting PD-L1 binding to PD-1 and B7.1, but does not disruptbinding of PD-1 to PD-L2. In some instances, the PD-L1 bindingantagonist antibody is MPDL3280A, which may be administered at a dose ofabout 700 mg to about 900 mg every two weeks (e.g., about 750 mg toabout 900 mg every two weeks, e.g., about 800 mg to about 850 mg everytwo weeks). In some embodiments, MPDL3280A is administered at a dose ofabout 840 mg every two weeks.

As a general proposition, the therapeutically effective amount of a PD-1axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., MPDL3280A)may be administered to a human will be in the range of about 0.01 toabout 50 mg/kg of patient body weight whether by one or moreadministrations. In some embodiments, for example, the antagonist (e.g.,anti-PD-L1 antibody, e.g., MPDL3280A) is administered in a dose of about0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 toabout 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 toabout 1 mg/kg administered daily, for example. In some embodiments, theantagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) is administeredat 15 mg/kg. However, other dosage regimens may be useful. In oneembodiment, a PD-1 axis binding antagonist (e.g., anti-PD-L1 antibody,e.g., MPDL3280A) is administered to a human at a dose of about 100 mg,about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg,about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg,about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg. In someembodiments, a PD-1 axis binding antagonist (e.g., anti-PD-L1 antibody,e.g., MPDL3280A) is administered at a dose of about 800 mg to about 850mg every two weeks. In some embodiments, a PD-1 axis binding antagonist(e.g., anti-PD-L1 antibody, e.g., MPDL3280A) is administered at a doseof about 840 mg every two weeks. The dose may be administered as asingle dose or as multiple doses (e.g., 2 or 3 doses), such asinfusions. The dose of the antibody administered in a combinationtreatment may be reduced as compared to a single treatment. In someembodiments, for example, the method for treating or delayingprogression of locally advanced or metastatic breast cancer in anindividual comprises a dosing regimen comprising treatment cycles,wherein the individual is administered, on days 1 and 15 of each cycle,a human PD-1 axis binding antagonist (e.g., anti-PD-L1 antibody, e.g.,MPDL3280A) at a dose of about 840 mg, wherein each cycle is 28 days(i.e., each cycle is repeated every 28 days). The progress of thistherapy is easily monitored by conventional techniques.

In some instances, the methods provided herein include administration ofan effective amount of a taxane (e.g., nab-paclitaxel (ABRAXANE®),paclitaxel, or docetaxel). In some instances, the taxane isnab-paclitaxel (ABRAXANE®). In some instances, the nab-paclitaxel(ABRAXANE®) is administered to the individual at a dose of about 100mg/m² to about 125 mg/m² every week. In some instances, thenab-paclitaxel (ABRAXANE®) is administered to the individual at a doseof about 100 mg/m² every week. As a general proposition, thetherapeutically effective amount of a taxane (e.g., nab-paclitaxel(ABRAXANE®)) administered to a human will be in the range of about 25 toabout 300 mg/m² (e.g., about 25 mg/m², about 50 mg/m², about 75 mg/m²,about 100 mg/m², about 125 mg/m², about 150 mg/m², about 175 mg/m²,about 200 mg/m², about 225 mg/m², about 250 mg/m², about 275 mg/m², orabout 300 mg/m²), whether by one or more administrations. For example,in some embodiments, about 100 mg/m² of nab-paclitaxel (ABRAXANE®) isadministered. In some embodiments, nab-paclitaxel (ABRAXANE®) isadministered at 100 mg/m² once a week. In some embodiments, about 125mg/m² of paclitaxel is administered. In some embodiments, paclitaxel isadministered at 200 mg/m² every three weeks. In some embodiments, thetaxane (e.g., nab-paclitaxel (ABRAXANE®)) may be administered weekly,every 2 weeks, every 3 weeks, every 4 weeks, on days 1, 8 and 15 of each21-day cycle, or on days 1, 8, and 15 of each 28-day cycle.

In some instances, the PD-1 axis binding antagonist (e.g., an anti-PD-L1antibody, e.g., MPDL3280A) and the taxane (e.g., nab-paclitaxel(ABRAXANE®)) are administered in a single dosing regimen. Theadministration of these agents may be concurrent or separate within thecontext of the dosing regimen. For example, in some instances, themethods provided herein include a dosing regimen comprising treatmentcycles, wherein the individual is administered, on days 1 and 15 of eachcycle, a human PD-1 axis binding antagonist at a dose of about 840 mg,and on days 1, 8, and 15 of each cycle, a taxane at a dose of about 100mg/m², each cycle being repeated every 28 days.

In some embodiments, the methods further comprise administering aneffective amount of a chemotherapeutic agent. In some instances, thechemotherapeutic agent is a platinum-based chemotherapeutic agent, suchas carboplatin.

In some embodiments, the individual is a human. In some embodiments, theindividual is suffering from locally advanced or metastatic breastcancer. In some embodiments, the metastatic breast cancer is mTNBC. Insome embodiments, the individual has had two or fewer prior cytotoxictreatment regimens for locally advanced or metastatic breast cancer. Insome embodiments, the individual has never had prior targeted systemictreatment for locally advanced or metastatic breast cancer. Thus, incertain instances, the methods for treating or delaying progression oflocally advanced or metastatic breast cancer (e.g., mTNBC) in anindividual or for enhancing immune function in an individual having alocally advanced or metastatic breast cancer can serve as a first-linetherapy for the individual.

In some embodiments, the individual has been treated with a cancertherapy before the combination treatment with a PD-1 axis bindingantagonist and a taxane. In some embodiments, the individual has cancerthat is resistant to one or more cancer therapies. In some embodiments,resistance to cancer therapy includes recurrence of cancer or refractorycancer. Recurrence may refer to the reappearance of cancer, in theoriginal site or a new site, after treatment. In some embodiments,resistance to a cancer therapy includes progression of the cancer duringtreatment with the anti-cancer therapy. In some embodiments, resistanceto a cancer therapy includes cancer that does not response to treatment.The cancer may be resistant at the beginning of treatment or it maybecome resistant during treatment. In some embodiments, the cancer is atearly stage or at late stage.

In some embodiments, the combination therapy of the invention comprisesadministration of a PD-1 axis binding antagonist and a taxane. The PD-1axis binding antagonist and the taxane (e.g., nab-paclitaxel(ABRAXANE®)) may be administered in any suitable manner known in theart. For example, the PD-1 axis binding antagonist and the taxane may beadministered sequentially (at different times) or concurrently (at thesame time). In some embodiments, the PD-1 axis binding antagonist is ina separate composition as the taxane. In some embodiments, the PD-1 axisbinding antagonist is in the same composition as the taxane.

The PD-1 axis binding antagonist and the taxane may be administered bythe same route of administration or by different routes ofadministration. In some embodiments, the PD-1 axis binding antagonist isadministered intravenously, intramuscularly, subcutaneously, topically,orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally. In some embodiments, the taxane is administeredintravenously, intramuscularly, subcutaneously, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally. Aneffective amount of the PD-1 axis binding antagonist and the taxane maybe administered for prevention or treatment of disease. The appropriatedosage of the PD-1 axis binding antagonist and/or the taxane may bedetermined based on the type of disease to be treated, the type of thePD-1 axis binding antagonist and the taxane, the severity and course ofthe disease, the clinical condition of the individual, the individual'sclinical history and response to the treatment, and the discretion ofthe attending physician.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery (e.g.,lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy,viral therapy, RNA therapy, immunotherapy, bone marrow transplantation,nanotherapy, monoclonal antibody therapy, or a combination of theforegoing. The additional therapy may be in the form of adjuvant orneoadjuvant therapy. In some embodiments, the additional therapy is theadministration of small molecule enzymatic inhibitor or anti-metastaticagent. In some embodiments, the additional therapy is the administrationof side-effect limiting agents (e.g., agents intended to lessen theoccurrence and/or severity of side effects of treatment, such asanti-nausea agents, etc.). In some embodiments, the additional therapyis radiation therapy. In some embodiments, the additional therapy issurgery. In some embodiments, the additional therapy is a combination ofradiation therapy and surgery. In some embodiments, the additionaltherapy is gamma irradiation. In some embodiments, the additionaltherapy is therapy targeting PI3K/AKT/mTOR pathway, HSP90 inhibitor,tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.The additional therapy may be one or more of the chemotherapeutic agentsdescribed herein.

In some embodiments, the methods further comprise administering aplatinum-based chemotherapeutic agent with the PD-1 axis bindingantagonist and taxane. In some embodiments, the platinum-basedchemotherapeutic agent is carboplatin. Dosages and administration ofcarboplatin are well-known in the art. An exemplary dosage ofcarboplatin is administered with a target area under the curve (AUC) of6 mg/ml. In some embodiments, the carboplatin is administeredintravenously every 3 weeks.

In some embodiments, the methods include treating an individualsuffering from mTNBC by administering anti-PD-L1 antibody MPDL3280A at800 mg IV administered every two weeks (q2w), along with nab-paclitaxel(ABRAXANE®) at 125 mg/m² IV every week (q1w), for three weeks within thecontext of a 4-week (28-day) treatment cycle, which may be repeateduntil there is loss of clinical benefit, complete response, remission,or otherwise, at the discretion of the attending physician.

V. Other Combination Therapies

Also provided herein are methods for treating or delaying progression oflocally advanced or metastatic breast cancer in an individual comprisingadministering to the individual a human PD-1 axis binding antagonist(e.g., anti-PD-L1 antibody, e.g., MPDL3280A) and a taxane (e.g.,nab-paclitaxel (ABRAXANE®)) in conjunction with another anti-canceragent or cancer therapy. In some embodiments, the methods compriseadministering to the individual a human PD-1 axis binding antagonist, ataxane, and an additional therapeutic agent.

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a chemotherapy or chemotherapeuticagent. In some embodiments, a PD-1 axis binding antagonist and a taxanemay be administered in conjunction with a radiation therapy orradiotherapeutic agent. In some embodiments, a PD-1 axis bindingantagonist and a taxane may be administered in conjunction with atargeted therapy or targeted therapeutic agent. In some embodiments, aPD-1 axis binding antagonist and a taxane may be administered inconjunction with an immunotherapy or immunotherapeutic agent, forexample a monoclonal antibody.

Without wishing to be bound to theory, it is thought that enhancing Tcell stimulation, by promoting an activating co-stimulatory molecule orby inhibiting a negative co-stimulatory molecule, may promote tumor celldeath thereby treating or delaying progression of cancer. In someembodiments, a PD-1 axis binding antagonist (e.g., anti-PD-L1 antibody,e.g., MPDL3280A) and a taxane (e.g., nab-paclitaxel (ABRAXANE®)) may beadministered in conjunction with an agonist directed against anactivating co-stimulatory molecule. In some embodiments, an activatingco-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR,CD137, CD27, HVEM, or CD127. In some embodiments, the agonist directedagainst an activating co-stimulatory molecule is an agonist antibodythat binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, orCD127. In some embodiments, a PD-1 axis binding antagonist (e.g.,anti-PD-L1 antibody, e.g., MPDL3280A) and a taxane (e.g., nab-paclitaxel(ABRAXANE®)) may be administered in conjunction with an antagonistdirected against an inhibitory co-stimulatory molecule. In someembodiments, an inhibitory co-stimulatory molecule may include CTLA-4(also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4,IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonistdirected against an inhibitory co-stimulatory molecule is an antagonistantibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3,B7-H4, IDO, TIGIT, MICA/B, or arginase.

In some embodiments, a PD-1 axis binding antagonist (e.g., anti-PD-L1antibody, e.g., MPDL3280A) and a taxane (e.g., nab-paclitaxel(ABRAXANE®)) may be administered in conjunction with an antagonistdirected against CTLA-4 (also known as CD152), for example, a blockingantibody. In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with ipilimumab (also known asMDX-010, MDX-101, or YERVOY®). In some embodiments, a PD-1 axis bindingantagonist and a taxane may be administered in conjunction withtremelimumab (also known as ticilimumab or CP-675,206). In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an antagonist directed against B7-H3(also known as CD276), for example, a blocking antibody. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with MGA271. In some embodiments, a PD-1axis binding antagonist and a taxane may be administered in conjunctionwith an antagonist directed against a TGF beta, for example, metelimumab(also known as CAT-192), fresolimumab (also known as GC1008), orLY2157299.

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a treatment comprising adoptivetransfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing achimeric antigen receptor (CAR). In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction witha treatment comprising adoptive transfer of a T cell comprising adominant-negative TGF beta receptor, e.g, a dominant-negative TGF betatype II receptor. In some embodiments, a PD-1 axis binding antagonistand a taxane may be administered in conjunction with a treatmentcomprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov IdentifierNCT00889954).

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an agonist directed against CD137 (alsoknown as TNFRSF9, 4-1 BB, or ILA), for example, an activating antibody.In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with urelumab (also known as BMS-663513). Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an agonist directed against CD40, forexample, an activating antibody. In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction withCP-870893. In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with an agonist directedagainst OX40 (also known as CD134), for example, an activating antibody.In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an anti-OX40 antibody (e.g., AgonOX).In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an agonist directed against CD27, forexample, an activating antibody. In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction withCDX-1127. In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with an antagonist directedagainst indoleamine-2,3-dioxygenase (IDO). In some embodiments, with theIDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an antibody-drug conjugate. In someembodiments, the antibody-drug conjugate comprises mertansine ormonomethyl auristatin E (MMAE). In some embodiments, a PD-1 axis bindingantagonist and a taxane may be administered in conjunction with andanti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599).In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with trastuzumab emtansine (also known asT-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech). In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with DMUC5754A. In some embodiments, a PD-1axis binding antagonist and a taxane may be administered in conjunctionwith an antibody-drug conjugate targeting the endothelin B receptor(EDNBR), for example, an antibody directed against EDNBR conjugated withMMAE.

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an angiogenesis inhibitor. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an antibody directed against a VEGF,for example, VEGF-A. In some embodiments, a PD-1 axis binding antagonist(e.g., anti-PD-L1 antibody, e.g., MPDL3280A) and a taxane (e.g.,nab-paclitaxel (ABRAXANE®)) may be administered in conjunction withbevacizumab (also known as AVASTIN®, Genentech). In some embodiments, aPD-1 axis binding antagonist and a taxane may be administered inconjunction with an antibody directed against angiopoietin 2 (also knownas Ang2). In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with MEDI3617.

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an antineoplastic agent. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an agent targeting CSF-1R (also knownas M-CSFR or CD115). In some embodiments, a PD-1 axis binding antagonistand a taxane may be administered in conjunction with anti-CSF-1R (alsoknown as IMC-CS4). In some embodiments, a PD-1 axis binding antagonistand a taxane may be administered in conjunction with an interferon, forexample interferon alpha or interferon gamma. In some embodiments, aPD-1 axis binding antagonist and a taxane may be administered inconjunction with Roferon-A (also known as recombinant Interferonalpha-2a). In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with GM-CSF (also known asrecombinant human granulocyte macrophage colony stimulating factor, rhuGM-CSF, sargramostim, or LEUKINE®). In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction withIL-2 (also known as aldesleukin or PROLEUKIN®). In some embodiments, aPD-1 axis binding antagonist and a taxane may be administered inconjunction with IL-12. In some embodiments, a PD-1 axis bindingantagonist and a taxane may be administered in conjunction with anantibody targeting CD20. In some embodiments, the antibody targetingCD20 is obinutuzumab (also known as GA101 or GAZYVA®) or rituximab. Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an antibody targeting GITR. In someembodiments, the antibody targeting GITR is TRX518.

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a cancer vaccine. In some embodiments,the cancer vaccine is a peptide cancer vaccine, which in someembodiments is a personalized peptide vaccine. In some embodiments thepeptide cancer vaccine is a multivalent long peptide, a multi-peptide, apeptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cellvaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an adjuvant. In some embodiments, aPD-1 axis binding antagonist and a taxane may be administered inconjunction with a treatment comprising a TLR agonist, for example,Poly-ICLC (also known as HILTONOL®), LPS, MPL, or CpG ODN. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with tumor necrosis factor (TNF) alpha. Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with IL-1. In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction withHMGB1. In some embodiments, a PD-1 axis binding antagonist and a taxanemay be administered in conjunction with an IL-10 antagonist. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an IL-4 antagonist. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an IL-13 antagonist. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an HVEM antagonist. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an ICOS agonist, e.g., byadministration of ICOS-L, or an agonistic antibody directed againstICOS. In some embodiments, a PD-1 axis binding antagonist and a taxanemay be administered in conjunction with a treatment targeting CX3CL1. Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a treatment targeting CXCL9. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a treatment targeting CXCL10. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a treatment targeting CCLS. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an LFA-1 or ICAM1 agonist. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a Selectin agonist.

In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with a targeted therapy. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an inhibitor of B-Raf. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with vemurafenib (also known as ZELBORAF®).In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with dabrafenib (also known as TAFINLAR®).In some embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with erlotinib (also known as TARCEVA®). Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an inhibitor of a MEK, such as MEK1(also known as MAP2K1) or MEK2 (also known as MAP2K2). In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with cobimetinib (also known as GDC-0973 orXL-518). In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with trametinib (also known asMEKINIST®). In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with an inhibitor of K-Ras. Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an inhibitor of c-Met. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with onartuzumab (also known as MetMAb). Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an inhibitor of Alk. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with AF802 (also known as CH5424802 oralectinib). In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with an inhibitor of aphosphatidylinositol 3-kinase (PI3K). In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction withBKM120. In some embodiments, a PD-1 axis binding antagonist and a taxanemay be administered in conjunction with idelalisib (also known asGS-1101 or CAL-101). In some embodiments, a PD-1 axis binding antagonistand a taxane may be administered in conjunction with perifosine (alsoknown as KRX-0401). In some embodiments, a PD-1 axis binding antagonistand a taxane may be administered in conjunction with an inhibitor of anAkt. In some embodiments, a PD-1 axis binding antagonist may beadministered in conjunction with MK2206. In some embodiments, a PD-1axis binding antagonist and a taxane may be administered in conjunctionwith GSK690693. In some embodiments, a PD-1 axis binding antagonist anda taxane may be administered in conjunction with GDC-0941. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with an inhibitor of mTOR. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with sirolimus (also known as rapamycin). Insome embodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with temsirolimus (also known as CCI-779 orTORISEL®). In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with everolimus (also known asRAD001). In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with ridaforolimus (also knownas AP-23573, MK-8669, or deforolimus). In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction withOSI-027. In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with AZD8055. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with INK128. In some embodiments, a PD-1axis binding antagonist and a taxane may be administered in conjunctionwith a dual PI3K/mTOR inhibitor. In some embodiments, a PD-1 axisbinding antagonist and a taxane may be administered in conjunction withXL765. In some embodiments, a PD-1 axis binding antagonist and a taxanemay be administered in conjunction with GDC-0980. In some embodiments, aPD-1 axis binding antagonist and a taxane may be administered inconjunction with BEZ235 (also known as NVP-BEZ235). In some embodiments,a PD-1 axis binding antagonist and a taxane may be administered inconjunction with BGT226. In some embodiments, a PD-1 axis bindingantagonist and a taxane may be administered in conjunction withGSK2126458. In some embodiments, a PD-1 axis binding antagonist and ataxane may be administered in conjunction with PF-04691502. In someembodiments, a PD-1 axis binding antagonist and a taxane may beadministered in conjunction with PF-05212384 (also known as PKI-587).

VI. Articles of Manufacture or Kits

In another embodiment of the invention, an article of manufacture or akit is provided comprising a PD-1 axis binding antagonist (e.g.,anti-PD-L1 antibody, e.g., MPDL3280A) and/or a taxane (e.g.,nab-paclitaxel (ABRAXANE®)). In some embodiments, the article ofmanufacture or kit further comprises package insert comprisinginstructions for using the PD-1 axis binding antagonist in conjunctionwith a taxane to treat or delay progression of locally advanced ormetastatic breast cancer in an individual or to enhance immune functionof an individual having locally advanced or metastatic breast cancer.Any of the PD-1 axis binding antagonist and/or taxanes described hereinmay be included in the article of manufacture or kits.

In some embodiments, the PD-1 axis binding antagonist and the taxane arein the same container or separate containers. Suitable containersinclude, for example, bottles, vials, bags and syringes. The containermay be formed from a variety of materials such as glass, plastic (suchas polyvinyl chloride or polyolefin), or metal alloy (such as stainlesssteel or hastelloy). In some embodiments, the container holds theformulation and the label on, or associated with, the container mayindicate directions for use. The article of manufacture or kit mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use. In someembodiments, the article of manufacture further includes one or more ofanother agent (e.g., a chemotherapeutic agent, and anti-neoplasticagent). Suitable containers for the one or more agent include, forexample, bottles, vials, bags and syringes.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

Examples

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Combination Treatment with Anti-PD-L1 Antibody andNab-Paclitaxel (ABRAXANE®) Achieved Response in a Phase Ib ClinicalTrial for Patients with Metastatic Triple-Negative Breast Cancer (mTNBC)

Metastatic triple-negative breast cancer (mTNBC) is associated with poorprognosis and is characterized by a high mutation rate, increased levelsof tumor-infiltrating lymphocytes, and high PD-L1 expression levels.MPDL3280A is a humanized monoclonal antibody that can restoretumor-specific T-cell immunity by inhibiting the binding of PD-L1 toPD-1 and has demonstrated durable responses as a monotherapy in mTNBC.This study is the first combination trial of a checkpoint inhibitor withchemotherapy in patients with mTNBC.

Methods

This arm of a multi-center, multi-arm phase Ib study evaluated MPDL3280Ain combination with weekly nab-paclitaxel in patients with mTNBC.Primary endpoints were safety and tolerability, with secondary endpointsof PK and clinical activity. Key eligibility criteria includedmeasurable disease; Eastern Cooperative Oncology Group (ECOG)performance status of 0 or 1; and 2 prior cytotoxic regimens. Patientsreceived atezolizumab 800 mg once every two weeks (on days 1 and 15)with nab-paclitaxel 125 mg/m² weekly (on days 1, 8, and 15) for 3 weeksin 4-week cycles, continued until loss of clinical benefit. Ifnab-paclitaxel was discontinued due to toxicity, MPDL3280A could becontinued as monotherapy. ORR was assessed by Response EvaluationCriteria In Solid Tumors (RECIST) v1.1. PD-L1 expression was scored at 4diagnostic levels based on PD-L1 staining on tumor cells andtumor-infiltrating immune cells in an immunohistochemical (IHC) assay.

Results

Eleven patients were safety-evaluable. All patients were female with amedian age of 58 years (age range: 32-75). No unexpected ordose-limiting toxicities were observed. The median duration of safetyfollow-up was 88 days (range: 27-182 days). The efficacy-evaluablepopulation consisted of 5 patients who had 1 scan and 3 monthsfollow-up. Of the five patients, four patients showed a partial response(PR) and one patient showed stable disease (SD). The observed resultsindicate that the combination of MPDL3280A and nab-paclitaxel is bothsafe and efficacious in patients with mTNBC.

Example 2: A Combination Treatment Regimen of Anti-PD-L1 Antibody andNab-Paclitaxel (ABRAXANE®) as a First-Line Therapy for Patients withmTNBC

As an alternative, a combination treatment regimen of MPDL3280A andnab-paclitaxel can serve as a first-line therapy for patients withmTNBC.

Patients with histologically documented locally advanced or metastaticTNBC; no prior systemic therapy for advanced TNBC; ECOG performancestatus of 0 or 1; and measurable disease per RECIST v1.1 may be dosedwith MPDL3280A (840 mg) on days 1 and 15, plus nab-paclitaxel (100mg/m²) on days 1, 8, and 15. All treatments are given on a 28-day cycle.Patients may be stratified by the presence of liver metastases, priortaxane therapy, and the PD-L1 status of tumor-infiltrating immune cells(IC0 vs IC1/2/3), which can be centrally evaluated by IHC. To capturepseudo-progression and delayed responses to MPDL3280A, patients withradiographic progression may continue to receive open-label MPDL3280Aalone or with nab-paclitaxel until unacceptable toxicity or loss ofclinical benefit.

OTHER EMBODIMENTS

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1-164. (canceled)
 165. A method for treating or delaying progression oflocally advanced or metastatic triple-negative breast cancer (TNBC) inan individual comprising administering to the individual an effectiveamount of atezolizumab and nab-paclitaxel.
 166. The method of claim 165,wherein the atezolizumab is administered to the individual at a dose ofabout 800 mg to about 850 mg every two weeks.
 167. The method of claim166, wherein the atezolizumab is administered to the individual at adose of about 840 mg every two weeks.
 168. The method of claim 165,wherein the TNBC is metastatic TNBC.
 169. The method of claim 165,wherein the cancer cells in the individual express PD-L1.
 170. Themethod of claim 165, wherein: (i) the individual has had two or fewerprior cytotoxic treatment regimens for locally advanced or metastaticbreast cancer; (ii) the individual has never had prior targeted systemictreatment for locally advanced or metastatic breast cancer; or (iii) thelocally advanced or metastatic TNBC is unresectable.
 171. The method ofclaim 165, wherein the nab-paclitaxel is administered before theatezolizumab, simultaneous with the atezolizumab, or after theatezolizumab.
 172. The method of claim 165, wherein the nab-paclitaxelis administered to the individual at a dose of about 100 mg/m² to about125 mg/m² every week.
 173. The method of claim 172, wherein thenab-paclitaxel is administered to the individual at a dose of about 100mg/m² every week.
 174. The method of claim 165, wherein the atezolizumaband/or the nab-paclitaxel are administered intravenously.
 175. Themethod of claim 165, further comprising administering to the individualan effective amount of a chemotherapeutic agent.
 176. A method ofenhancing immune function in an individual having locally advanced ormetastatic TNBC comprising administering to the individual an effectiveamount of atezolizumab and nab-paclitaxel.
 177. The method of claim 176,wherein: (i) CD8+ T cells in the individual have enhanced priming,activation, proliferation, and/or cytolytic activity relative to priorto the administration of the atezolizumab and the nab-paclitaxel; (ii)the number of CD8+ T cells is elevated relative to prior toadministration of the atezolizumab and the nab-paclitaxel; (iii) Tregfunction is suppressed relative to prior to the administration of theatezolizumab and the nab-paclitaxel; and/or (iv) T cell exhaustion isdecreased relative to prior to the administration of the atezolizumaband the nab-paclitaxel.
 178. The method of claim 176, whereinatezolizumab is administered to the individual at a dose of about 800 mgto about 850 mg every two weeks.
 179. The method of claim 178, whereinatezolizumab is administered to the individual at a dose of about 840 mgevery two weeks.
 180. The method of claim 176, wherein the TNBC ismetastatic TNBC.
 181. The method of claim 176, wherein the cancer cellsin the individual express PD-L1.
 182. The method of claim 176, wherein:(i) the individual has had two or fewer prior cytotoxic treatmentregimens for locally advanced or metastatic breast cancer; (ii) theindividual has never had prior targeted systemic treatment for locallyadvanced or metastatic breast cancer; or (iii) the locally advanced ormetastatic TNBC is unresectable.
 183. The method of claim 176, whereinthe nab-paclitaxel is administered to the individual at a dose of about100 mg/m² to about 125 mg/m² every week.
 184. The method of claim 183,wherein the nab-paclitaxel is administered to the individual at a doseof about 100 mg/m² every week.
 185. The method of claim 176, wherein theatezolizumab and/or the nab-paclitaxel are administered intravenously.186. The method of claim 176, further comprising administering to theindividual an effective amount of a chemotherapeutic agent.